DPP7 Antibody

What is DPP7 and what is its biological significance?

DPP7 is a post-proline-cleaving aminopeptidase primarily expressed in quiescent lymphocytes. It plays a crucial role in the regulation of lymphocyte quiescence and immune regulation. When lymphocytes are stimulated by antigens, they may mobilize from their quiescent state to divide, and inhibition of DPP7 can result in apoptosis of resting (but not activated) lymphocytes . DPP7 belongs to the family of prolyl peptidases, which are enzymes characterized by their ability to cleave proline-containing peptides. The DPP family includes several members such as dipeptidyl peptidase 4, DPP7, DPP8, DPP9, prolyl endopeptidase, prolyl endopeptidase-like, and fibroblast activation protein .

How does DPP7 expression differ between normal and cancerous tissues?

Expression analysis shows that DPP7 is significantly upregulated in colorectal cancer tissues compared to adjacent non-tumor tissues. This upregulation pattern extends beyond colorectal cancer to numerous other cancer types, including adrenocortical carcinoma, bladder urothelial carcinoma, breast invasive carcinoma, cervical carcinoma, and many others as demonstrated through TCGA database analysis . Specifically in colorectal cancer, high mRNA expression of DPP7 was observed in approximately 5% of colon cancer samples according to CbioPortal and TCGA database analysis . Immunohistochemical validation using clinical samples has confirmed this differential expression pattern .

What are the common methods for detecting DPP7 expression?

Several complementary techniques can be employed to detect and quantify DPP7 expression:

• Quantitative Real-Time PCR (qRT-PCR): For mRNA expression analysis using specific primers. Published primers include:

  • GAPDH (control): Forward 5′-GGAGTCAACGGATTTGGTCGT-3′, Reverse 5′-TCTCGCTCCTGGAAGATGGT-3′

  • DPP7: Forward 5′-GAAGCGTTCCGACAGATCAAG-3′, Reverse 5′-TCAGGTCCTTCTCGTCTGACA-3′

• Immunohistochemistry (IHC): For protein expression in tissue samples using specific antibodies such as DPP7 antibody (NBP1-84986, Novusbio) or DPP7 antibody (AP61032, abcepta) .

• Western Blotting: For protein expression analysis in cell lysates using anti-DPP7 antibodies with appropriate secondary antibodies (typically anti-rabbit for DPP7) .

What are the key considerations for DPP7 antibody selection?

When selecting a DPP7 antibody for research, consider:

• Antibody Type: Polyclonal antibodies (like rabbit polyclonal to DPP7) offer high sensitivity but may have more background compared to monoclonal antibodies .

• Epitope Location: Some antibodies target specific regions (such as the C-terminal region of human DPP7), which may affect their performance in different applications .

• Validated Applications: Confirm the antibody has been validated for your specific application (IHC, Western blot, etc.) in publications.

• Species Reactivity: Ensure the antibody recognizes DPP7 in your species of interest.

• Purification Method: Antibodies purified by immunogen affinity chromatography often exhibit higher specificity .

How does DPP7 expression correlate with clinical outcomes in cancer patients?

Multiple studies have established that high DPP7 expression correlates with poor prognosis in colorectal cancer patients. The subgroup analyses of both colon and rectal cancer confirmed that patients with high DPP7 expression had worse prognosis compared to those with low expression . This prognostic significance appears to be particularly pronounced in patients with positive lymph node metastasis (N1 & N2) .

The relationship between DPP7 and prognosis varies across different cancer types. While high DPP7 expression is associated with poor prognosis in colorectal cancer and chronic lymphocytic leukemia, studies have interestingly found that it correlates with better prognosis in breast cancer patients, suggesting tissue-specific functions of DPP7 in different cancers .

RNA Interference (RNAi) for DPP7 Knockdown:

Effective siRNA sequences targeting DPP7 include:

• siRNA-1: GAAGCGTTCCGACAGATCA

• siRNA-2: CGTCTGGACCACTTCAACT

For stable knockdown, shRNA with the sequence CGTCTGGACCACTTCAACT in a pLV3-U6-MCS-shRNA-EF1a-CopGFP-Puro vector has been successfully employed .

Cell Proliferation Assays:

To assess the impact of DPP7 on cancer cell proliferation:

• CCK-8 Assay: Seed 3,000 cells per well in 96-well plates after DPP7 knockdown/overexpression, culture for 24-96 hours, and measure absorbance at 450nm after 2-hour incubation with CCK-8 solution .

• EDU Assay: Seed 10,000 cells per well in 96-well plates, allow growth to normal phase, then perform staining according to kit instructions and observe under an inverted fluorescence microscope .

Migration and Invasion Assays:

Transwell assays with or without Matrigel coating can effectively measure the impact of DPP7 modulation on cancer cell migration and invasion capabilities .

How does DPP7 influence tumor immune microenvironment?

DPP7 has been shown to impact the tumor immune microenvironment in several ways:

• Immune Cell Infiltration: Single-sample Gene Set Enrichment Analysis (ssGSEA) reveals correlations between DPP7 expression and the infiltration of various immune cells, including B cells, CD8+ T cells, T helper cells, cytotoxic cells, natural killer cells, dendritic cells, regulatory T cells, and macrophages .

• Immune Checkpoint Correlation: DPP7 expression shows significant correlation with immune checkpoint genes, including programmed cell death 1 (PD-1/PDCD1), programmed cell death ligand 1 (PD-L1/CD274), and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) .

• Immune Evasion: Overexpression of DPP7 suppresses the immune cells' cytotoxic function against tumors in the tumor microenvironment, as demonstrated through co-culture experiments of tumor cells with immune cells .

What are the optimal parameters for immunohistochemical detection of DPP7?

For optimal IHC detection of DPP7 in colorectal cancer samples:

• Antibody Selection: Both DPP7 antibody (NBP1-84986, Novusbio) and DPP7 antibody (AP61032, abcepta) have been successfully used at dilutions of 1:300 .

• Tissue Preparation: Paraffin-embedded tissue blocks should be sectioned and processed according to standard protocols. For comparison studies, it's advantageous to include both tumor tissue and adjacent normal tissue (ideally collected at least 10cm from the tumor margin) .

• Controls: Include positive and negative controls to validate staining specificity. Colorectal cancer tissue microarrays can be valuable for standardized multi-sample analysis .

• Scoring System: Implement a standardized scoring system that accounts for both staining intensity and percentage of positive cells to ensure reproducible quantification of DPP7 expression levels .

How can I design co-culture experiments to study DPP7's impact on immune cell function?

To investigate how DPP7 expression in cancer cells affects immune cell function:

• Cell Preparation:

  • Generate DPP7-overexpressing and DPP7-knockdown colorectal cancer cell lines using appropriate vectors and controls.

  • Isolate immune cells (T cells, NK cells, etc.) from peripheral blood or prepare immune cell lines.

• Co-Culture Setup:

  • Direct co-culture: Mix cancer cells with immune cells at appropriate ratios.

  • Indirect co-culture: Use transwell systems to study effects mediated by soluble factors without direct cell contact.

• Functional Assays:

  • Cytotoxicity assays to measure immune cell-mediated killing of cancer cells.

  • Cytokine production analysis using ELISA or flow cytometry.

  • Immune checkpoint expression analysis on both cancer cells and immune cells.

• Analysis Methods:

  • Flow cytometry to assess immune cell activation markers and cancer cell death.

  • Microscopy to visualize immune cell-cancer cell interactions.

  • Gene expression analysis of both cell populations after co-culture .

How can I validate the specificity of a DPP7 antibody for my experiments?

To ensure antibody specificity:

• Positive and Negative Controls:

  • Use tissues/cells known to express DPP7 as positive controls.

  • Include DPP7-knockout or siRNA-treated samples as negative controls.

• Blocking Peptide Experiments: Pre-incubate the antibody with its immunizing peptide before application to confirm signal specificity.

• Multiple Detection Methods: Validate expression using complementary techniques (IHC, Western blot, qRT-PCR) to confirm consistency.

• Multiple Antibodies: When possible, use different antibodies targeting distinct epitopes of DPP7 to confirm findings.

• Molecular Weight Verification: For Western blotting, confirm that the detected band matches the expected molecular weight of DPP7.

What are the key pathways and mechanisms influenced by DPP7 in cancer progression?

Research indicates that DPP7 influences cancer progression through several mechanisms:

• Cell Proliferation: DPP7 enhances cancer cell proliferation as demonstrated through CCK-8 and EDU assays on colorectal cancer cell lines .

• Epithelial-Mesenchymal Transition (EMT): DPP7 may influence EMT markers including E-cadherin, N-cadherin, and Vimentin, affecting cancer cell invasiveness .

• Signaling Pathway Modulation: Gene Set Enrichment Analysis (GSEA) has identified several signaling pathways modulated by DPP7 overexpression, including:

  • Neuroactive ligand-receptor interaction

  • Olfactory transduction signaling

• Immune Evasion: DPP7 promotes immune evasion by suppressing cytotoxic immune cell function in the tumor microenvironment .

• Drug Response Correlation: Analysis using Genomics of Drug Sensitivity in Cancer (GDSC) and Genomics of Therapeutics Response Portal (CTRP) databases has revealed correlations between DPP7 expression and response to certain cancer therapeutics .

How can DPP7 be utilized as a prognostic biomarker in clinical research?

DPP7 shows significant potential as a prognostic biomarker:

• Patient Stratification: High vs. low DPP7 expression can stratify colorectal cancer patients into different prognostic groups, particularly those with lymph node metastasis .

• Multi-marker Panels: Combining DPP7 with other biomarkers may enhance prognostic accuracy. Researchers should consider integrating DPP7 into existing biomarker panels.

• Liquid Biopsy Potential: Investigating DPP7 expression in circulating tumor cells or cell-free DNA might provide less invasive prognostic information.

• Treatment Response Prediction: The correlation between DPP7 expression and drug sensitivity suggests potential for predicting treatment response, which warrants further investigation .

What are promising research directions for targeting DPP7 therapeutically?

Several approaches for therapeutic targeting of DPP7 show promise:

• DPP7 Inhibitors: Development of specific DPP7 inhibitors may induce apoptosis in cancer cells, as inhibition of DPP7 has been shown to increase apoptosis in various cancer cells .

• Immune Checkpoint Combination: Given the correlation between DPP7 expression and immune checkpoint markers, combination strategies targeting both DPP7 and immune checkpoints may enhance treatment efficacy .

• siRNA-based Therapeutics: The successful use of siRNA for DPP7 knockdown in experimental settings suggests potential for RNA interference-based therapeutic approaches .

• Antibody-Drug Conjugates: DPP7 antibodies could potentially be developed into antibody-drug conjugates for targeted therapy of DPP7-overexpressing tumors.

• Cancer Subtype-specific Approaches: Given the tissue-specific functions of DPP7 across different cancers, therapeutic strategies should be tailored to specific cancer types .

What are common challenges in DPP7 antibody-based experiments and how can they be addressed?

• Nonspecific Binding:

  • Optimize antibody concentration (1:300 dilution has been effective for IHC)

  • Include appropriate blocking steps with bovine serum albumin or serum

  • Increase washing duration and frequency

• Variability in IHC Staining:

  • Standardize tissue processing and antigen retrieval methods

  • Use automated staining platforms when available

  • Implement positive and negative controls for each experiment

• Western Blot Issues:

  • Ensure proper sample preparation and protein denaturation

  • Use freshly prepared buffers and reagents

  • Optimize transfer conditions for proteins in DPP7's molecular weight range

• qRT-PCR Optimization:

  • Validate primer specificity using melt curve analysis

  • Include appropriate housekeeping genes (GAPDH has been successfully used)

  • Perform efficiency tests for accurate quantification

How should researchers interpret contradictory findings regarding DPP7 across different cancer types?

When faced with contradictory findings regarding DPP7 across different cancer types:

• Acknowledge Tissue Specificity: DPP7 appears to have tissue-specific functions, with high expression associated with poor prognosis in colorectal cancer but better prognosis in breast cancer .

• Consider Cellular Context: The role of DPP7 may depend on the specific cellular context, including the presence of other molecular alterations and the tumor microenvironment.

• Examine Methodological Differences: Contradictions may arise from differences in experimental methods, antibodies used, scoring systems, or cutoff values for defining "high" versus "low" expression.

• Analyze Patient Cohorts: Differences in patient demographics, disease stage, or treatment history might explain discrepancies between studies.

• Investigate Isoforms or Post-translational Modifications: Different antibodies may detect specific isoforms or post-translational modifications of DPP7, potentially explaining contradictory results.