CSF3R Antibody

What is CSF3R and what is its biological function?

CSF3R (Colony Stimulating Factor 3 Receptor), also known as G-CSF receptor (G-CSFR), functions as the receptor for granulocyte colony-stimulating factor (CSF3). This receptor plays a crucial role in the proliferation, differentiation, and survival of cells along the neutrophilic lineage. CSF3R is essential for granulocytic maturation and may additionally function in certain adhesion or recognition events at the cell surface . The receptor has been detected in myelogenous leukemia cell line KG-1, leukemia U-937 cell line, bone marrow cells, placenta, and peripheral blood granulocytes, with specific isoforms showing differential expression patterns across tissues .

What are the structural characteristics and protein specifications of CSF3R?

CSF3R has a calculated molecular weight of 92,156 Da, with the commercially available antibodies often targeting the central region (amino acids 252-280) of the human protein . The protein contains multiple functional domains important for ligand binding and signal transduction. Key specifications include:

What experimental applications are most suitable for CSF3R antibodies?

CSF3R antibodies can be utilized across multiple experimental platforms with specific optimization requirements for each application:

For long-term storage, maintain the antibody refrigerated at 2-8°C for up to 2 weeks, or store at -20°C in small aliquots to prevent freeze-thaw cycles which can compromise antibody integrity .

How can researchers validate the specificity of CSF3R antibodies?

When validating CSF3R antibodies, researchers should implement a multi-faceted approach:

• Conduct epitope mapping confirmation to verify binding to the targeted region (e.g., amino acids 252-280 in the central region of human CSF3R)

• Perform blocking experiments using the immunizing peptide (KLH conjugated synthetic peptide)

• Include positive control tissues known to express CSF3R (bone marrow, placenta)

• Implement negative controls lacking the primary antibody

• Compare staining patterns across multiple antibodies targeting different CSF3R epitopes

Discrepancies in staining patterns may indicate non-specific binding or cross-reactivity issues that should be resolved before proceeding with critical experiments.

How do CSF3R mutations contribute to leukemic progression?

CSF3R mutations, particularly truncation mutations that affect the distal cytoplasmic portion of the G-CSF receptor, confer a strong clonal advantage at the hematopoietic stem cell (HSC) level that depends on exogenous G-CSF. Research demonstrates these mutations lead to:

• Enhanced G-CSF-induced proliferation in hematopoietic stem cells

• Increased phosphorylation of Stat5 and transcription of Stat5 target genes

• Establishment of clonal dominance through inappropriate Stat5 activation

Comparative analysis shows that the area under the curve (AUC) for Stat5 activation differs significantly between wild-type (203 ± 73) and d715 G-CSFR mutant cells (771 ± 187; P = 0.05) in KSL cells, while Stat3 activation is attenuated in the mutants . This altered signaling profile likely contributes to the leukemogenic potential of cells harboring CSF3R mutations.

What methodologies should be used to detect low-frequency CSF3R mutations?

Detection of CSF3R mutations is highly dependent on the sensitivity of the method employed:

Ultra-deep sequencing of cDNA markedly increases sensitivity for identifying CSF3R mutant clones with minor allele frequencies (MAF) as low as 0.001, enabling detection of early pre-leukemic clones . This technical advantage explains why earlier studies using less sensitive methods reported significantly varying mutation frequencies.

What is the significance of specific CSF3R mutations in myeloid disorders?

Different CSF3R mutations carry varied significance in myeloid disorders:

• CSF3R T618I point mutation: Emerged as a key driver in chronic neutrophilic leukemia (CNL) and atypical chronic myeloid leukemia (aCML)

• CSF3R T640N mutation: Under investigation as a potential marker for CNL/aCML diagnosis and as a therapeutic target

• Truncation mutations of CSF3R: Present in approximately 40% of patients with severe congenital neutropenia (SCN) and strongly associated with progression to AML/MDS

Approximately 80% of congenital neutropenia patients who developed leukemia harbor acquired CSF3R mutations, with all leukemic cells in these patients affected by these mutations . This suggests CSF3R mutations are necessary but insufficient for leukemic transformation, requiring additional cooperative mutations.

How should O-glycosylation of CSF3R be effectively studied?

O-glycosylation of CSF3R can be studied using the following methodological approach:

• Transfect CSF3R constructs (WT, T618I, T640N) into 293T17 cells

• Culture in the presence of 50 μM GalNAz (Thermo Scientific) for 48 hours

• Wash cells and incubate in 1ml PBS with 1% FBS and 30 μM DBCO-sulfo-link-biotin conjugate for 1 hour

• Lyse cells in cell lysis buffer containing complete mini protease inhibitor tablets

• Incubate lysates overnight with streptavidin-agarose

• Subject precipitated biotinylated proteins to immunoblot analysis for CSF3R

This bioorthogonal labeling strategy allows specific detection of O-glycosylated CSF3R species, enabling comparison between wild-type and mutant receptors to identify alterations in glycosylation patterns that may contribute to pathogenesis.

What considerations are important when designing experiments to study CSF3R signaling pathways?

When investigating CSF3R signaling pathways, researchers should carefully design experiments that address:

• Differential activation kinetics: Studies show both magnitude and duration of Stat5 activation are enhanced in d715 G-CSFR mutant cells, while Stat3 activation is attenuated . Time-course experiments are therefore critical.

• Target gene expression analysis: Key genes differentially regulated by mutant CSF3R include Sprr2a, Tcrg, Pim2, Cdkn1a, Cish, and Socs2 . RNA-seq or targeted qPCR analysis of these genes provides valuable insights into altered signaling.

• Cell population selection: CSF3R signaling differs significantly between stem cells and more differentiated progenitors. KSL (c-Kit+ Sca-1+ Lin-) cells should be isolated for studying effects in stem cell populations .

• Genetic manipulation controls: Studies in myeloid progenitors lacking both Stat5A and Stat5B have demonstrated abrogation of the proliferative advantage conferred by mutant CSF3R, emphasizing the importance of appropriate genetic controls .

How can researchers differentiate between pathogenic CSF3R mutations and benign polymorphisms?

Differentiating pathogenic CSF3R mutations from benign polymorphisms requires a multi-dimensional approach:

• In silico prediction: Utilize multiple algorithms to predict the functional impact of variants. For example, the p.D510H SNP was predicted to have damaging effects by 4 out of 6 prediction algorithms, while p.D320N was predicted to be benign by all six algorithms despite both having similar minor allele frequencies .

• Functional assays: Test the effect of mutations on:

  • Cytokine-independent growth

  • Receptor dimerization

  • Stat3/Stat5 activation patterns

  • O-glycosylation modifications

  • Response to JAK inhibitors

• Domain location analysis: Mutations in conserved domains may have different consequences than those in variable regions. For instance, p.D320N located in the conserved cytokine receptor homology domain was predicted to be benign, while a novel p.M222T polymorphism in the fibronectin type III-like domain was predicted to have severe effects on protein function .

• Correlation with disease progression: Track the association between specific mutations and disease outcomes in longitudinal studies.

What approaches should be used to monitor CSF3R mutation acquisition and clonal expansion?

For effective monitoring of CSF3R mutation acquisition and clonal expansion:

• Sequential sampling: Implement annual monitoring of CSF3R mutations using deep sequencing to trace pre-leukemic clones over extended time periods .

• Quantitative assessment: Track changes in the minor allele frequency (MAF) of mutations as an indicator of clonal expansion.

• Multi-gene panel analysis: Include additional genes known to cooperate with CSF3R in leukemic transformation (e.g., RUNX1, ASXL1, SUZ12, EP300) .

• Therapeutic correlation: Monitor relationships between G-CSF therapy (dosage, duration) and emergence or expansion of CSF3R mutations. Current evidence suggests no correlation between CSF3R mutations and G-CSF dose required to achieve sufficient neutrophil counts .

How might single-cell sequencing advance our understanding of CSF3R mutations?

Single-cell sequencing technologies hold significant promise for CSF3R research by:

• Enabling detection of rare subclones with CSF3R mutations that may be masked in bulk analysis

• Revealing the co-occurrence of multiple mutations within individual cells

• Providing insights into the heterogeneity of CSF3R mutant populations

• Allowing trajectory analysis to map the evolution of pre-leukemic clones

• Identifying cell-specific signaling alterations induced by CSF3R mutations

These advantages could substantially improve early detection of pre-leukemic clones and enhance our understanding of clonal evolution in CSF3R-mutated disorders.

What therapeutic implications arise from improved CSF3R mutation detection?

Advanced CSF3R mutation detection methods have several important therapeutic implications:

• Early intervention strategies: The ability to detect low-frequency CSF3R mutations (MAF as low as 0.001) enables identification of patients at risk for leukemic transformation before clinical manifestation .

• Personalized therapy approaches: Different CSF3R mutations may exhibit variable sensitivity to targeted therapies, potentially enabling mutation-guided treatment selection.

• Modification of G-CSF protocols: Understanding how G-CSF treatment influences CSF3R mutation acquisition and expansion may guide modifications to treatment protocols that minimize leukemic risk.

• Combination therapy development: Identification of cooperating mutations enables rational design of combination therapies targeting multiple pathways involved in leukemic transformation.

• Monitoring treatment response: Sequential tracking of CSF3R mutation burden provides a potential biomarker for assessing response to therapy and early detection of relapse.