Circulating Tumor Cells: How Far Have We Come with Mining These Seeds of Metastasis?

Circulating tumor cells (CTCs) are cancer cells that slough off from the tumor and circulate in the peripheral blood and lymphatic system as micro metastases that eventually results in macro metastases. Through a simple blood draw, sensitive CTC detection from clinical samples has proven to be a useful tool for determining the prognosis of cancer. Recent technological developments now make it possible to detect CTCs reliably and repeatedly from a simple and straightforward blood test. Multicenter trials to assess the clinical value of CTCs have demonstrated the prognostic value of these cancer cells. Studies on CTCs have filled huge knowledge gap in understanding the process of metastasis since their identification in the late 19th century. However, these rare cancer cells have not been regularly used to tailor precision medicine and or identify novel druggable targets. In this review, we have attempted to summarize the milestones of CTC-based research from the time of identification to molecular characterization. Additionally, the need for a paradigm shift in dissecting these seeds of metastasis and the possible future avenues to improve CTC-based discoveries are also discussed.

Preclinical models to study patient-derived circulating tumor cells and metastasis.

Circulating tumor cells (CTCs) that are detached from the tumor can be precursors of metastasis. The majority of studies focus on enumeration of CTCs from patient blood to predict recurrence and therapy outcomes. Very few studies have managed to expand CTCs to investigate their functional dynamics with respect to genetic changes, tumorigenic potential, and response to drug treatment. A growing amount of evidence based on successful CTC expansion has revealed novel therapeutic targets that are associated with the process of metastasis. In this review, we summarize the successes, challenges, and limitations that collectively contribute to the better understanding of metastasis using patient-derived CTCs as blood-borne seeds of metastasis. The roadblocks and future avenues to move CTC-based scientific discoveries forward are also discussed.

Tumorigenic circulating tumor cells from xenograft mouse models of non-metastatic NSCLC patients reveal distinct single cell heterogeneity and drug responses.

BACKGROUND: Circulating tumor cells (CTCs) are liquid biopsies that represent micrometastatic disease and may offer unique insights into future recurrences in non-small cell lung cancer (NSCLC). Due to CTC rarity and limited stability, no stable CTC-derived xenograft (CDX) models have ever been generated from non-metastatic NSCLC patients directly. Alternative strategies are needed to molecularly characterize CTCs and means of potential future metastases in this potentially curable patient group. METHODS: Surgically resected NSCLC primary tumor tissues from non-metastatic patients were implanted subcutaneously in immunodeficient mice to establish primary tumor patient-derived xenograft (ptPDX) models. CTCs were isolated as liquid biopsies from the blood of ptPDX mice and re-implanted subcutaneously into naïve immunodeficient mice to generate liquid biopsy CTC-derived xenograft (CDX) tumor models. Single cell RNA sequencing was performed and validated in an external dataset of non-xenografted human NSCLC primary tumor and metastases tissues. Drug response testing in CDX models was performed with standard of care chemotherapy (carboplatin/paclitaxel). Blockade of MYC, which has a known role in drug resistance, was performed with a MYC/MAX dimerization inhibitor (10058-F4). RESULTS: Out of ten ptPDX, two (20%) stable liquid biopsy CDX mouse models were generated. Single cell RNA sequencing analysis revealed an additional regenerative alveolar epithelial type II (AT2)-like cell population in CDX tumors that was also identified in non-xenografted NSCLC patients' metastases tissues. Drug testing using these CDX models revealed different treatment responses to carboplatin/paclitaxel. MYC target genes and c-MYC protein were upregulated in the chemoresistant CDX model, while MYC/MAX dimerization blocking could overcome chemoresistance to carboplatin/paclitaxel. CONCLUSIONS: To overcome the lack of liquid biopsy CDX models from non-metastatic NSCLC patients, CDX models can be generated with CTCs from ptPDX models that were originally established from patients' primary tumors. Single cell analyses can identify distinct drug responses and cell heterogeneities in CDX tumors that can be validated in NSCLC metastases tissues. CDX models deserve further development and study to discover personalized strategies against micrometastases in non-metastatic NSCLC patients.

Epigenetic Regulation of Cancer Immune Cells.

The epigenetic regulation of immune response involves reversible and heritable changes that do not alter the DNA sequence. Though there have been extensive studies accomplished relating to epigenetic changes in cancer cells, recent focus has been shifted on epigenetic-mediated changes in the immune cells including T cells, Macrophages, Natural Killer cells and anti-tumor immune responses. This review compiles the most relevant and recent literature related to the role of epigenetic mechanisms including DNA methylation and histone modifications in immune cells of wide range of cancers. We also include recent research with respect to role of the most relevant transcription factors that epigenetically control the anti-tumor immune response. Finally, a statement of future direction that promises to look forward for strategies to improve immunotherapy in cancer.

Current and Prospective Methods for Assessing Anti-Tumor Immunity in Colorectal Cancer.

Colorectal cancer (CRC) remains one of the deadliest malignancies worldwide despite recent progress in treatment strategies. Though immune checkpoint inhibition has proven effective for a number of other tumors, it offers benefits in only a small group of CRC patients with high microsatellite instability. In general, heterogenous cell groups in the tumor microenvironment are considered as the major barrier for unveiling the causes of low immune response. Therefore, deconvolution of cellular components in highly heterogeneous microenvironments is crucial for understanding the immune contexture of cancer. In this review, we assimilate current knowledge and recent studies examining anti-tumor immunity in CRC. We also discuss the utilization of novel immune contexture assessment methods that have not been used in CRC research to date.

Circulating Giant Tumor-Macrophage Fusion Cells Are Independent Prognosticators in Patients With NSCLC.

INTRODUCTION: Various subtypes of circulating cancer-associated cells in the blood are described. A unique circulating, large, and polymorphonuclear cell with a dual epithelial and myeloid phenotype has been suggested as a tumor-macrophage fusion cell (TMF). The goal of the study was to identify the impact of distinct TMFs on survival among patients with NSCLC. METHODS: In this prospective trial, 7.5 mL of whole blood sample was collected. After microfilter enrichment, immunofluorescent staining was performed, identifying TMFs as greater than or equal to 30 µm in size and dual epithelial (cytokeratin 8, 18, or 19-, or epithelial cell adhesion molecule-positive) and myeloid- or macrophage-positive (CD14- or CD45-positive) cells with at least one 4',6-diamidino-2-phenylindole+ nucleus. RESULTS: Circulating TMFs were identified in 88 of 115 patients (76.5%) with NSCLC (mean 3.052 [SEM ± 0.306]; median 2 [range 0-17]) but were rare in long-term smokers without cancer (6 of 87 [6.9%]; 0.081 [±0.034]; 0 [0-2]), and absent in 20 healthy controls. Comparing the presence of TMFs in patients with NSCLC versus smokers without cancer, specificity was 93.1% (95% confidence interval: 85.6-97.4%) and sensitivity 76.5% (95% confidence interval: 67.7%-83.9%). TMF counts correlated with American Joint Committee on Cancer tumor stages. More importantly, more than one TMF and giant TMFs sizes greater than or equal to 50 µm were associated with statistically significantly shorter overall and cancer-specific disease-free (p < 0.05) survival after curative resection for stage I to IIIA. Giant TMFs greater than or equal to 50 µm size were an independent survival predictor by multivariate analysis. CONCLUSIONS: Circulating, in particular, giant TMFs are associated with aggressive clinical behavior in surgically treated patients with NSCLC. The biological role of unique TMFs will need to be further investigated, as these may have a potential impact on immune responses toward cancer.

Tumor-Cell-Macrophage Fusion Cells as Liquid Biomarkers and Tumor Enhancers in Cancer.

Although molecular mechanisms driving tumor progression have been extensively studied, the biological nature of the various populations of circulating tumor cells (CTCs) within the blood is still not well understood. Tumor cell fusion with immune cells is a longstanding hypothesis that has caught more attention in recent times. Specifically, fusion of tumor cells with macrophages might lead to the development of metastasis by acquiring features such as genetic and epigenetic heterogeneity, chemotherapeutic resistance, and immune tolerance. In addition to the traditional FDA-approved definition of a CTC (CD45-, EpCAM+, cytokeratins 8+, 18+ or 19+, with a DAPI+ nucleus), an additional circulating cell population has been identified as being potential fusions cells, characterized by distinct, large, polymorphonuclear cancer-associated cells with a dual epithelial and macrophage/myeloid phenotype. Artificial fusion of tumor cells with macrophages leads to migratory, invasive, and metastatic phenotypes. Further studies might investigate whether these have a potential impact on the immune response towards the cancer. In this review, the background, evidence, and potential relevance of tumor cell fusions with macrophages is discussed, along with the potential role of intercellular connections in their formation. Such fusion cells could be a key component in cancer metastasis, and therefore, evolve as a diagnostic and therapeutic target in cancer precision medicine.

Circulating tumor cell clusters are a potential biomarker for detection of non-small cell lung cancer.

OBJECTIVES: Circulating tumor cell (CTC) clusters (≥2 CTCs in aggregate) detected in the peripheral blood have predictive value in solid cancers, including non-small cell lung cancer (NSCLC). The goal of the study was to investigate the presence of CTC clusters in NSCLC patients and in high-risk screening subjects having no or benign nodules in a screening low-dose CT (LDCT). MATERIALS AND METHODS: In a prospective pilot trial, 7.5 ml peripheral blood was collected from treatment-naïve NSCLC patients, LDCT screening subjects (55-80 years, ≥30 pack-year smoking history) with no (Lung-RADS 1) or benign lung nodules (Lung-RADS 2), and healthy never-smoking controls. CTCs were enriched by size, also allowing CTC cluster isolation. For CTC identification and enumeration, immunofluorescence staining was performed for cytokeratins (CK) 8/18 and/or 19, EpCAM, CD45, and nuclei were stained with DAPI. Clinicopathological data were collected, and LDCT interpreted by the American College of Radiology Lung-RADS criteria. RESULTS: CTC clusters were detected in 12/29 (41.4%) of all NSCLC patients, but not found in 31 high-risk screening subjects with Lung-RADS 1 or Lung-RADS 2 (P < 0.05). Since non-clustered, single CTCs were detectable in both groups of NSCLC patients (100%) and in 18/31 (58.1%) of high-risk screening subjects. No CTCs were detected in 20 healthy control subjects. CONCLUSION: This pilot study suggests that CTC clusters are a useful and specific liquid biomarker to further explore for screening by LDCT and risk stratification of NSCLC patients. Future prospective studies with higher subject numbers will need to be performed.

Plant DNases are potent therapeutic agents against Echis carinatus venom-induced tissue necrosis in mice.

Echis carinatus envenomation leads to severe tissue necrosis at the bitten site by releasing DNA from immune cells that blocks the blood flow. An earlier report has shown that exogenous DNase 1 offers protection against such severe local tissue necrosis. Tricosanthus tricuspidata is a medicinal plant and the paste prepared from its leaves has been used extensively for the treatment of snakebite-induced tissue necrosis. Most studies including reports from our laboratory focused on plant secondary metabolite as therapeutic molecules against snakebite envenomation. However, the involvement of hydrolytic enzymes including DNase in treating snake venom-induced tissue necrosis has not been addressed. Several folk medicinal plants used against snakebite treatment showed the presence of DNase activity and found to be rich in T. tricuspidata. Further, purified T. tricuspidata DNase showed a single sharp peak in reversed-phase high-performance liquid chromatography (RP-HPLC) with an apparent molecular mass of 17 kDa. T. tricuspidata DNase exhibited potent DNA degrading activity performed using agarose gel electrophoresis, spectrophotometric assay, and DNA zymography. In addition, purified DNase from T. tricuspidata was able to neutralize E. carinatus venom-induced mouse tail tissue necrosis and normalized elevated serum creatine kinase (CK) and lactate dehydrogenase (LDH) levels 30 minutes post venom injection. T. tricuspidata DNase was also able to reverse E. carinatus venom-induced histopathological changes and collagen depletion in mice tail tissue. All these observed pharmacological actions of T. tricuspidata DNase were inhibited by sodium fluoride (NaF). This study provides scientific validation of the traditional use of T. tricuspidata leaf paste in the healing of snakebite-induced tissue necrosis and might be exploited to treat snake venom-induced local toxicity.