Keywords
salivary tumor cells - oral squamous cell carcinoma - noninvasive diagnostics - early
detection - liquid biopsy
Introduction
Oral squamous cell carcinoma (OSCC) accounts for a notable global health burden, with
a 5-year survival rate remaining at approximately 50% despite advancements in diagnosis
and treatment.[1] The primary factors contributing to poor outcomes are late-stage diagnosis, metastasis,
and recurrent disease. Effective diagnostic tools that facilitate early detection
and consistent disease monitoring are critical for improving prognosis. However, traditional
diagnostic approaches, including biopsies and blood-based tests, are invasive, expensive,
and unsuitable for frequent use.[2] In this context, saliva offers a novel, noninvasive medium for cancer diagnostics.[3] Saliva's accessibility, ease of collection, and continuous interaction with the
oral cavity make it an attractive alternative for identifying biomarkers associated
with oral cancers.[4] Among these biomarkers, tumor cells shed into saliva, referred to here as salivary
tumor cells (STCs), represent a transformative tool in the diagnosis and monitoring
of OSCC. This review examines the clinical potential of STCs, their detection methodologies,
and the associated challenges in harnessing this technology for routine cancer diagnostics.
While numerous studies have extensively investigated circulating tumor cells (CTCs)
in peripheral blood across various cancers, there is a notable lack of published data
specifically addressing the presence and diagnostic utility of tumor cells in saliva.
Therefore, this review draws upon the foundational understanding of CTC biology and
detection technologies as a reference point to explore the potential of detecting
tumor-derived cells in saliva. We propose the term STCs to differentiate these locally
shed cells from blood-borne CTCs, emphasizing their unique biological context within
the oral cavity. This conceptual framework aims to stimulate further clinical and
translational research in this emerging domain.
Revisiting Terminology: Why STCs?
Revisiting Terminology: Why STCs?
CTC is commonly used to describe cancer cells that enter the bloodstream and circulate
through the body.[5] However, in the context of saliva, these cells are not “circulating” but are primarily
exfoliated or shed from primary tumors in the oral cavity or surrounding regions.[6] Several mechanisms facilitate the entry of tumor cells into saliva. One route is
direct shedding, where tumor cells are exfoliated directly from the primary site into
the saliva.[7] Another is through exosomal release, wherein tumor-derived exosomes carrying specific
genetic or phenotypic markers are secreted into saliva.[8] Passive diffusion is also possible, allowing tumor cells to enter saliva as a result
of necrosis or other passive mechanisms. Additionally, in rare instances, translocation
may occur, where tumor cells migrate from the systemic circulation to the salivary
glands before appearing in saliva.[9]
To reflect this distinction and avoid confusion, alternative terminologies are proposed:
-
Tumor cells in saliva—: Directly describes their source without implying circulation
-
STCs: Captures tumor-derived biomarkers, including cells, deoxyribonucleic acid (DNA),
ribonucleic acid (RNA), and proteins
-
Detached or exfoliated tumor cells: Highlights their detachment from tumor masses
This review adopts the term STCs to encompass all tumor-derived cells detectable in
saliva.
STC Biology
STCs are likely to originate directly from primary oral lesions through mechanisms
such as passive exfoliation, active detachment, or alterations within the tumor microenvironment.[10]
[11] Their shedding is influenced by factors like epithelial disruption from invasive
growth, secretion of proteolytic enzymes (e.g., matrix metalloproteinases) that weaken
cell–matrix adhesion, the induction of epithelial–mesenchymal transition (EMT) facilitating
motility and resistance to cell death, and chronic inflammation that alters epithelial
turnover.[12]
[13]
[14] STCs are more likely to be released into saliva from ulcerated or exophytic lesions,
particularly in areas subject to mechanical friction or trauma. Once in saliva, tumor
cells face a hostile environment characterized by enzymatic degradation, antimicrobial
components such as secretory immunoglobulin A, fluctuating pH, and constant interaction
with the oral microbiota.[15]
[16] To survive these conditions, STCs may remain embedded in mucus, be encapsulated
within exosomes or microvesicles, and exhibit phenotypic adaptability—such as expressing
mucin-associated or EMT-related proteins—that enhances their resilience.[17]
[18]
CTC Survival Mechanisms: Insights for STCs
CTC Survival Mechanisms: Insights for STCs
Tumor cells to survive in harsh environments like the bloodstream exhibit the following
adaptive mechanisms: Immune evasion that is facilitated by the expression of proteins such as HER2 and PD-L1, which help
tumor cells escape immune detection and contribute to chemoresistance.[19] Additionally, platelet shielding occurs when tumor cells form aggregates with platelets, providing protection from
immune cells and shear forces.[20]
Genetic plasticity further supports tumor survival, as the high genetic diversity of tumor cells contributes
to both treatment resistance and increased metastatic potential.[21] These insights, though derived from studies on CTCs in blood, underline the importance
of targeted approaches to STC detection and analysis in saliva. [Tables 1] and [2] provide a comparative summary of CTC versus STC and serum-based CTC versus STC,
respectively.
Table 1
Comparison of CTCs and STCs
|
Feature
|
Circulating tumor cells (CTCs)
|
Salivary tumor cells (STCs)
|
|
Origin
|
Intravasation from primary/metastatic tumor into blood
|
Shedding from oral mucosal tumors directly into saliva
|
|
Microenvironment
|
Hemodynamically active, immune surveillance
|
Enzyme-rich, variable pH, mucosal immunity, microbiota -rich
|
|
Survival mechanisms
|
EMT, immune evasion
|
Mucus embedding, exosomal transport, EMT features
|
|
Metastatic role
|
Systemic dissemination and metastasis
|
Local invasion marker, possible lymphatic spread
|
|
Accessibility
|
Requires venipuncture, preprocessing
|
Noninvasive, simple saliva collection
|
|
Diagnostic utility
|
Established in many cancers (e.g., breast, lung, prostate)
|
Emerging concept in oral cancers
|
|
Challenges
|
CTCs are extremely rare, often fewer than 10 cells/mL of blood amidst millions of
normal blood cells. This low abundance requires ultrasensitive detection technologies,
increasing cost and complexity
CTCs undergo mechanical and oxidative stress while circulating, making them fragile
and susceptible to lysis or loss of viability during collection and processing[22]
CTC detection is often biased toward epithelial markers (e.g., EpCAM). Tumor cells
undergoing epithelial–mesenchymal transition (EMT) may downregulate these markers,
leading to false negatives[23]
|
High degradation risk, epithelial contamination which reduces specificity and makes
it difficult to distinguish malignant cells from benign ones.
Saliva's microbes and mucins can interfere with staining and molecular tests, leading
to unclear or
false results[24]
|
Abbreviations: EpCAM, epithelial cell adhesion molecule; EMT, epithelial–mesenchymal
transition.
Table 2
Comparative analysis STCs vs. serum-based CTCs
|
Variable
|
Serum-based CTCs
|
STCs
|
|
Collection
|
Venipuncture (invasive)
|
Saliva sample (noninvasive)
|
|
Concentration
|
Higher in systemic cancers
|
Lower; localized to oral cancers
|
|
Clinical insights
|
Systemic tumor spread
|
Local tumor progression
|
|
Ease of access
|
Moderate
|
High
|
|
Cost
|
Higher
|
Lower
|
Abbreviations: CTC, circulating tumor cell; STC, salivary tumor cell.
Clinical Applications of STC Detection
Clinical Applications of STC Detection
Saliva-based diagnostics hold immense potential for noninvasive cancer care. Some
of the applications include: They can serve as biomarkers for early-stage OSCC, potentially
reducing diagnostic delays. STCs also enable frequent monitoring of tumor progression
and response to therapy. Additionally, molecular profiling of STCs supports personalized
treatment strategies tailored to individual patients. Moreover, saliva collection
proves particularly valuable for screening in high-risk populations, especially in
low-resource settings or for large-scale cancer screening programs.
Isolation Techniques
Efficient isolation of STCs is critical due to their low abundance in saliva compared
to other cellular components. Several advanced methods have been developed to enhance
specificity and sensitivity:
-
Filtration:
Filtration-based systems operate on the principle of separating cells based on size. Tumor cells, being larger
than most other cells and debris in saliva, are retained while smaller components
pass through the filter. The technology involves the use of specialized membrane filters
with defined pore sizes (e.g., 8–10 µm) to effectively capture tumor cells.
-
Immunomagnetic separation:
-
Immunomagnetic separation is based on the principle of using magnetic beads coated with antibodies specific
to epithelial or tumor markers such as EpCAM (epithelial cell adhesion molecule).
These beads selectively bind to tumor cells, which can then be isolated magnetically.[25] The technology employs advanced bead designs and antibody conjugation to ensure
high affinity and specificity. After separation, the captured cells can be released
for downstream analysis using gentle enzymatic or chemical treatments.
-
Advantages:
-
Limitations:
-
✓ Dependency on marker expression, can vary across tumor types
-
✓ Expensive due to use of specialized reagents
-
Microfluidic devices:
-
Lab-on-chip devices operate on the principle of leveraging the physical and biochemical properties of
tumor cells for automated isolation. These devices utilize microscale channels and
chambers to selectively capture cells based on size, deformability, or surface markers.
The technology includes integrated systems with optical sensors for real-time monitoring,
and advanced designs that allow for parallel processing of multiple samples.
-
Advantages:
-
Limitations:
Characterization Approaches
Characterization Approaches
Once isolated, STCs undergo detailed characterization to confirm their malignancy
and to derive clinically relevant insights. These approaches utilize cutting-edge
imaging and molecular biology techniques:
-
Morphological analysis:
-
Methodology: Tumor cells exhibit unique morphological features such as: enlarged nuclei with
irregular shapes, high nuclear-to-cytoplasmic ratio, prominent nucleoli, and mitotic
figures, indicating active cell division[26]
-
Tools required: Light microscopy, phase-contrast microscopy, or fluorescence microscopy with specific
dyes to enhance visualization and imaging software for automated analysis of cellular
features
-
Clinical
utility: Provides quick confirmation of malignancy based on visual criteria
-
Limitations: Morphological overlap with nontumor cells can lead to false positives, requiring
additional molecular confirmation
-
Molecular profiling:
-
Techniques:
-
✓ Reverse transcription-polymerase chain reaction: Amplifies specific tumor-associated messenger RNA or DNA markers, such as mutations
in TP53 or human papillomavirus DNA
-
✓ Fluorescence in situ hybridization: Detects chromosomal abnormalities and specific genetic alterations (e.g., amplifications
or deletions) in individual cells[27]
-
Advantages:
-
Applications:
-
Flow cytometry:
-
Principle: Tumor cells are labeled with fluorescent antibodies targeting tumor- specific markers
(e.g., EpCAM, cytokeratins, or PD-L1). As the cells pass through a laser beam, fluorescence
intensity is measured to identify and quantify the tumor cells[28]
-
Technology:
-
Advantages:
-
✓ Rapid and high throughput
-
✓ Quantitative, providing precise cell counts
-
✓ Can distinguish between live and dead cells using viability dyes
-
Limitations:
Emerging Technologies in STC Detection
Emerging Technologies in STC Detection
In addition to the above methods, ongoing advancements aim to overcome existing challenges.
Single-cell sequencing provides unparalleled insights into the genetic and epigenetic landscape of individual
tumor cells, aiding in the identification of novel biomarkers and therapeutic targets.[29]
Artificial intelligence (AI)-based image analysis allows the integration of AI in microscopy and imaging, enabling automated, accurate
classification of tumor cells and reducing observer bias. Nanotechnology is also being employed, with nanoparticle-based systems developed to enhance the
sensitivity of detection techniques, particularly for rare tumor cells in saliva.[30] These innovations promise to make STC detection more robust, accessible, and clinically
impactful, paving the way for their integration into routine cancer diagnostic.
Challenges
Detecting and processing STCs present multiple technical and biological challenges.
The extremely low concentration of tumor cells in saliva, especially in early-stage
cancers, makes detection difficult, while their heterogeneous nature complicates the
development of standardized identification protocols. Additionally, the high risk
of microbial contamination and the presence of nontumor components such as epithelial
cells and debris in saliva can pose significant challenges for culturing and accurate
analysis, requiring the use of antimicrobial agents. The fragile nature of tumor cells
and their biomarkers in saliva can affect the stability and reliability of analysis,
while the lack of validated, sensitive, and specific detection tools continues to
limit clinical applicability. Overall, significant standardization and technological
advancement are needed before saliva-based tumor cell diagnostics can be reliably
integrated into routine clinical practice.
Strengths
STCs have key advantages over CTCs, especially for oral cancer. STCs can be collected
noninvasively through saliva, improving patient comfort and compliance. They are cost-effective,
easy to repeat, and suitable for large-scale screening. Unlike CTCs, STCs reflect
local tumor activity, allowing earlier detection of oral lesions. Their potential
for real-time, localized monitoring positions STCs as a promising tool in oral cancer
diagnostics, despite the need for further validation and standardization.
Gray Areas
STCs face limitations due to the lack of standardized detection methods and variability
in saliva composition. Low cell yield, contamination, and degradation make isolation
difficult. STC diagnostics are still experimental, with limited validation and accuracy
compared to CTCs, requiring further research.
Generalizability
The generalizability of STCs as diagnostic markers remains limited. Without broader
validation across diverse populations and cancer types, STCs cannot yet be reliably
applied as a universal diagnostic tool.
Conclusion
STCs represent a paradigm shift in noninvasive cancer diagnostics, offering a simple
and patient-friendly alternative for detecting and monitoring oral cancers. Despite
challenges such as low abundance and heterogeneity, ongoing advancements in detection
technologies and molecular analysis are steadily enhancing their feasibility and reliability.
By addressing current limitations through innovation and validation studies, saliva-based
tumor cell diagnostics could transform cancer care, making it more accessible, personalized,
and effective.
