ccdc85ca Antibody

What is CCDC85A and what experimental approaches are used to study it?

CCDC85A is a human protein encoded by the CCDC85A gene (UniProt accession: Q96JN8), characterized by coiled-coil motifs that mediate protein interactions. It belongs to the delta-interacting protein A (DIPA) family, which comprises CCDC85A, CCDC85B, and CCDC85C, each containing a pair of conserved coiled-coil motifs but with less homology in their C-terminal sequences .

For experimental approaches, researchers typically employ:

• Immunohistochemistry (IHC) using validated antibodies (1:200–1:500 dilution)

• Cell-based assays with cancer cell lines (U-87MG, Capan-1)

• CRISPR-Cas9 gene editing to create knockout models

• Lentiviral infection for stable expression of CCDC85A cDNA

• Co-immunoprecipitation assays to study protein-protein interactions

In knockout studies, researchers have successfully targeted CCDC85A using CRISPR-Cas9 in Capan1 cells, with sgRNA sequences designed using prediction tools like GeneArt CRISPR Search .

What are the optimal conditions for using CCDC85A antibodies in immunohistochemistry?

When conducting immunohistochemical studies with CCDC85A antibodies, researchers should consider the following protocol parameters:

The antibody has been validated through rigorous protocols including staining of 44 normal human tissues and 20 cancer types, making it suitable for comprehensive tissue analysis.

How should researchers validate the specificity of CCDC85A antibodies?

Validation of CCDC85A antibodies should follow a multi-step approach:

• Western blot analysis: Confirm a single band at the expected molecular weight (~45 kDa)

• Immunohistochemistry controls:

  • Positive control tissues with known CCDC85A expression

  • Negative controls using isotype-matched irrelevant antibodies

  • Absorption controls using immunizing peptide

• Genetic validation: Test antibody in CCDC85A knockout models created via CRISPR-Cas9

• Orthogonal validation: Compare results from antibody-based detection with mRNA expression

• Cross-reactivity assessment: Test against related family members (CCDC85B and CCDC85C)

For CRISPR-based validation, researchers should amplify the target DNA fragment, design sgRNAs using prediction tools, and confirm successful targeting by in vitro digestion of the target genomic DNA with the sgRNA and Cas9 nuclease .

How does CCDC85A regulate endoplasmic reticulum stress responses?

CCDC85A plays a crucial role in modulating cellular responses to endoplasmic reticulum (ER) stress through the following mechanism:

• CCDC85A directly associates with molecular chaperones GRP78 and GRP94

• This interaction disrupts the binding of these chaperones to PERK (PKR-like ER kinase)

• The displacement of negative regulators leads to sustained activation of PERK upon ER stress

• Activated PERK phosphorylates eIF2α, which attenuates eIF2α-mediated global translation

• This leads to upregulation of ATF4, a transcription factor that enhances cell survival under stress conditions

This molecular pathway represents a self-guard cellular response against ER stress. Through these interactions, CCDC85A essentially acts as a positive regulator of the unfolded protein response (UPR), allowing cells to better cope with ER stress conditions that would otherwise lead to apoptosis .

What is the relationship between CCDC85A and chemoresistance in cancer models?

CCDC85A contributes significantly to chemoresistance in cancer through several mechanisms:

Experimental evidence demonstrates that injection of NF-derived exosomes containing miR-224-3p into xenograft tumors increases tumor shrinkage during cisplatin treatment, highlighting a potential therapeutic approach .

How do microRNAs regulate CCDC85A expression in cancer microenvironments?

MicroRNA-mediated regulation of CCDC85A represents a critical mechanism in tumor-stromal interactions:

• miR-224-3p as a direct regulator: Research has identified miR-224-3p as a key regulator that targets CCDC85A mRNA, suppressing its expression .

• Fibroblast-derived exosomal delivery: Normal fibroblasts (NFs) secrete exosomes containing miR-224-3p, which are taken up by cancer cells. This exosomal transfer of miR-224-3p leads to downregulation of CCDC85A in cancer cells, making them more susceptible to cisplatin-induced apoptosis .

• Cancer-associated fibroblast dysfunction: When normal fibroblasts transform into cancer-associated fibroblasts (CAFs), the expression of miR-224-3p is reduced. This results in diminished exosomal transfer of miR-224-3p to cancer cells, allowing for increased CCDC85A expression and consequently enhanced chemoresistance .

• Therapeutic potential: Experimental evidence has shown that direct injection of NF-derived exosomes containing miR-224-3p into xenograft tumors increases tumor shrinkage during cisplatin treatment, suggesting a potential therapeutic approach .

This microRNA-mediated regulation represents a novel mechanism for how the tumor microenvironment influences cancer cell behavior and treatment response.

What genetic modification techniques are most effective for studying CCDC85A function?

Researchers investigating CCDC85A function have successfully employed several genetic modification approaches:

• CRISPR-Cas9 gene targeting:

  • Design sgRNA sequences using prediction tools such as GeneArt CRISPR Search

  • Amplify T7 promoter and sgRNA template sequences with long linker primers

  • Transcribe sgRNAs using MEGA T7 transcription kit

  • Validate sgRNA efficiency by in vitro digestion of target DNA before cellular genome editing

  • Deliver Cas9 protein and sgRNA to target cells (e.g., Capan1) for knockout generation

• Lentiviral overexpression:

  • Clone CCDC85A cDNA into lentiviral expression vectors

  • Co-transfect with packaging vectors into HEK293T cells for viral particle production

  • Infect target cells and select using appropriate antibiotics (e.g., puromycin)

  • Isolate and expand single colonies to establish stable cell lines

• miRNA-based regulation:

  • Transfect cells with miR-224-3p mimics or inhibitors to modulate CCDC85A expression

  • Alternatively, isolate exosomes from normal fibroblasts containing miR-224-3p for treatment of cancer cells

  • Measure changes in CCDC85A expression and downstream effects on cell phenotype

These approaches can be used independently or in combination to comprehensively investigate CCDC85A function in various cellular contexts.

How can researchers effectively study CCDC85A protein interactions and their functional consequences?

To investigate CCDC85A protein interactions and their functional significance, researchers should employ these methodological approaches:

• Co-immunoprecipitation (Co-IP) assays:

  • Use anti-CCDC85A antibody to pull down protein complexes

  • Analyze interacting partners (e.g., GRP78, GRP94) by Western blotting or mass spectrometry

  • Perform reciprocal Co-IP with antibodies against suspected binding partners

  • Include appropriate controls (IgG, lysate input)

• Proximity ligation assays (PLA):

  • Visualize protein-protein interactions in situ

  • Particularly useful for studying CCDC85A interactions with ER stress components (GRP78, GRP94, PERK)

  • Quantify interaction signals across different experimental conditions

• Domain mapping experiments:

  • Generate truncated CCDC85A constructs lacking specific domains

  • Express these constructs in cells and assess their interaction with binding partners

  • Identify critical domains for protein-protein interactions and function

• Functional consequence assessment:

  • Monitor the activation status of the PERK pathway (p-PERK, p-eIF2α, ATF4)

  • Measure ER stress responses using UPR reporter assays

  • Evaluate cell survival under ER stress conditions (e.g., tunicamycin, thapsigargin treatment)

  • Assess chemosensitivity using cell viability assays following cisplatin treatment

These approaches provide complementary information about CCDC85A interactions and their impact on cellular phenotypes, particularly in the context of ER stress and chemoresistance.

What experimental designs best assess CCDC85A's role in cancer cell migration and invasion?

To rigorously evaluate CCDC85A's impact on cancer cell migration and invasion, researchers should implement the following experimental approaches:

• 2D migration assays:

  • Wound healing/scratch assays with CCDC85A-overexpressing and knockout cells

  • Time-lapse microscopy to track cell movement dynamics

  • Quantify wound closure rates under different conditions (e.g., ER stress inducers)

• Transwell migration and invasion assays:

  • Use Boyden chambers with or without Matrigel coating

  • Compare migration/invasion between CCDC85A-manipulated cells

  • Assess the impact of miR-224-3p treatment on CCDC85A-mediated effects

• 3D spheroid invasion models:

  • Generate spheroids from cancer cells with varying CCDC85A expression

  • Embed spheroids in extracellular matrix (e.g., Matrigel, collagen)

  • Monitor invasion into surrounding matrix over time

  • Analyze differences in invasion patterns and distances

• Molecular pathway analysis:

  • Examine expression of epithelial-mesenchymal transition (EMT) markers

  • Assess cytoskeletal reorganization via immunofluorescence

  • Evaluate matrix metalloproteinase (MMP) expression and activity

  • Investigate potential crosstalk between CCDC85A-mediated ER stress responses and migration/invasion pathways

• In vivo metastasis models:

  • Inject CCDC85A-modified cancer cells into appropriate animal models

  • Track metastatic spread using imaging techniques

  • Correlate metastatic potential with CCDC85A expression levels

  • Test the effect of exosomal miR-224-3p delivery on metastasis

These complementary approaches provide a comprehensive assessment of CCDC85A's role in cancer cell migration and invasion, which has been documented in glioblastoma (U-87MG) and pancreatic cancer (Capan-1) cell lines.

How can CCDC85A antibodies be utilized in patient-derived cancer samples?

CCDC85A antibodies offer valuable tools for analyzing patient-derived cancer samples through multiple approaches:

• Tissue microarray (TMA) analysis:

  • Apply validated CCDC85A antibodies (1:200–1:500 dilution) to TMAs containing multiple patient samples

  • Evaluate expression patterns across different cancer types and grades

  • Correlate expression with clinical parameters and outcomes

  • The antibody has been validated on 44 normal human tissues and 20 cancer types, making it reliable for such applications

• Prognostic biomarker evaluation:

  • Assess CCDC85A expression in tumor tissues versus matched normal tissues

  • Correlate expression levels with patient survival and treatment response

  • Particularly relevant for gastric cancer (scirrhous subtypes) and pancreatic adenocarcinoma, where CCDC85A upregulation has been associated with poor prognosis

• Cancer-associated fibroblast (CAF) analysis:

  • Examine CCDC85A expression in the tumor microenvironment, particularly in CAFs

  • Compare with normal fibroblasts from the same patient

  • Assess correlation with miR-224-3p levels in these cell populations

• Patient-derived xenograft (PDX) models:

  • Establish PDX models from patient tumors

  • Monitor CCDC85A expression during treatment with cisplatin or other chemotherapeutics

  • Test the effect of miR-224-3p-containing exosomes on treatment response

  • Evaluate tumor shrinkage in response to combined therapy approaches

These applications can provide valuable insights into the clinical relevance of CCDC85A in human cancers and potentially identify patient subgroups that might benefit from therapies targeting CCDC85A-related pathways.

What methodologies should be used to investigate CCDC85A as a potential therapeutic target?

To evaluate CCDC85A as a therapeutic target, researchers should employ a systematic approach combining multiple methodologies:

• Target validation strategies:

  • CRISPR-Cas9 knockout or siRNA knockdown in multiple cancer cell lines

  • Phenotypic assessment of cell proliferation, survival, migration, and chemosensitivity

  • Rescue experiments to confirm specificity of observed effects

  • In vivo tumor growth and metastasis studies using genetic models

• Molecular mechanism characterization:

  • Detailed analysis of CCDC85A interactions with GRP78 and GRP94

  • Mapping of critical binding domains and identification of potential druggable pockets

  • Evaluation of downstream effects on PERK/eIF2α/ATF4 pathway activation

  • Assessment of combined inhibition of CCDC85A and other ER stress modulators

• Therapeutic approach development:

  • miR-224-3p delivery strategies (e.g., nanoparticles, exosomes) to target CCDC85A

  • Combination studies with cisplatin or other chemotherapeutics that induce ER stress

  • Development of peptides or small molecules that disrupt CCDC85A interactions with GRP78/GRP94

  • Testing in patient-derived organoids and xenograft models

• Biomarker identification:

  • Develop assays to measure CCDC85A expression/activity in patient samples

  • Identify patient subgroups most likely to benefit from CCDC85A-targeted therapy

  • Establish correlation between CCDC85A levels and response to ER stress-inducing treatments

This comprehensive approach would provide strong evidence for the potential of CCDC85A as a therapeutic target, particularly in cancers where CCDC85A contributes to chemoresistance through modulation of ER stress responses.

How can single-cell analysis techniques advance our understanding of CCDC85A function?

Single-cell approaches offer powerful new insights into CCDC85A biology that cannot be captured by bulk tissue analysis:

• Single-cell RNA sequencing (scRNA-seq):

  • Profile CCDC85A expression across heterogeneous cell populations within tumors

  • Identify specific cell types that express high levels of CCDC85A

  • Correlate CCDC85A expression with cell states (proliferative, invasive, stress-resistant)

  • Map co-expression patterns with ER stress response genes

  • Compare expression in cancer cells versus stromal components, particularly normal fibroblasts versus CAFs

• Single-cell protein analysis:

  • Use mass cytometry (CyTOF) with anti-CCDC85A antibodies

  • Simultaneously detect CCDC85A with markers of ER stress (phospho-PERK, phospho-eIF2α)

  • Analyze protein expression at the single-cell level in heterogeneous tumor samples

• Spatial transcriptomics/proteomics:

  • Map CCDC85A expression patterns within the tumor microenvironment

  • Correlate spatial distribution with miR-224-3p expression

  • Identify regional variations in CCDC85A levels relative to tumor architecture

  • Assess relationship between CCDC85A expression and proximity to fibroblasts or other stromal cells

• Live-cell imaging techniques:

  • Generate fluorescently tagged CCDC85A constructs

  • Track subcellular localization and dynamics during ER stress responses

  • Monitor interactions with GRP78/GRP94 in real-time using FRET-based approaches

  • Analyze single-cell behavior in migration and invasion assays

These advanced single-cell approaches would provide unprecedented resolution of CCDC85A function in heterogeneous cancer tissues and could reveal novel aspects of its role in tumor biology.

What contradictions exist in the current understanding of CCDC85A function, and how might they be resolved?

Several important contradictions and knowledge gaps exist in our understanding of CCDC85A function that warrant further investigation:

Addressing these contradictions would significantly advance our understanding of CCDC85A biology and its potential as a therapeutic target.