Peripheral blood MФ–cancer cell fusion hybrids identified in cancer patients correlate with disease stage and overall survival.

Metastatic progression defines the final stages of tumor evolution and underlies the majority of cancer-related deaths. The heterogeneity in disseminated tumor cell populations capable of seeding and growing in distant organ sites contributes to the development of treatment resistant disease. We recently reported the identification of a novel tumor-derived cell population, circulating hybrid cells (CHCs), harboring attributes from both macrophages and neoplastic cells, including functional characteristics important to metastatic spread. These disseminated hybrids outnumber conventionally defined circulating tumor cells (CTCs) in cancer patients. It is unknown if CHCs represent a generalized cancer mechanism for cell dissemination, or if this population is relevant to the metastatic cascade. Herein, we detect CHCs in the peripheral blood of patients with cancer in myriad disease sites encompassing epithelial and non-epithelial malignancies. Further, we demonstrate that in vivo-derived hybrid cells harbor tumor-initiating capacity in murine cancer models and that CHCs from human breast cancer patients express stem cell antigens, features consistent with the potential to seed and grow at metastatic sites. Finally, we reveal heterogeneity of CHC phenotypes reflect key tumor features, including oncogenic mutations and functional protein expression. Importantly, this novel population of disseminated neoplastic cells opens a new area in cancer biology and renewed opportunity for battling metastatic disease.

Background: Uveal melanoma is an aggressive cancer with high metastatic risk. Recently, we identified a circulating cancer cell population that co-expresses neoplastic and leukocyte antigens, termed circulating hybrid cells (CHCs). In other cancers, CHCs are more numerous and better predict oncologic outcomes compared to circulating tumor cells (CTCs). We sought to investigate the potential of CHCs as a prognostic biomarker in uveal melanoma. Methods: We isolated peripheral blood monocular cells from uveal melanoma patients at the time of primary treatment and used antibodies against leukocyte and melanoma markers to identify and enumerate CHCs and CTCs by immunocytochemistry. Results: Using a multi-marker approach to capture the heterogeneous disseminated tumor cell population, detection of CHCs was highly sensitive in uveal melanoma patients regardless of disease stage. CHCs were detected in 100% of stage I-III uveal melanoma patients (entire cohort, n = 68), whereas CTCs were detected in 58.8% of patients. CHCs were detected at levels statically higher than CTCs across all stages (p = 0.05). Moreover, CHC levels, but not CTCs, predicted 3 year progression-free survival (p < 0.03) and overall survival (p < 0.04). Conclusion: CHCs are a novel and promising prognostic biomarker in uveal melanoma.

Continual renewal of the intestinal epithelium is dependent on active- and slow-cycling stem cells that are confined to the crypt base. Tight regulation of these stem cell populations maintains homeostasis by balancing proliferation and differentiation to support critical intestinal functions. The hierarchical relation of discrete stem cell populations in homeostasis or during regenerative epithelial repair remains controversial. Although recent studies have supported a model for the active-cycling leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5)+ intestinal stem cell (ISC) functioning upstream of the slow-cycling B lymphoma Mo-MLV insertion region 1 homolog (Bmi1)-expressing cell, other studies have reported the opposite relation. Tools that facilitate simultaneous analyses of these populations are required to evaluate their coordinated function. We used novel monoclonal antibodies (mAbs) raised against murine intestinal epithelial cells in conjunction with ISC-green fluorescent protein (GFP) reporter mice to analyze relations between ISC populations by microscopy. Ex vivo 3-dimensional cultures, flow cytometry, and quantitative reverse-transcription polymerase chain reaction analyses were performed. Two novel mAbs recognized distinct subpopulations of the intestinal epithelium and when used in combination permitted isolation of discrete Lgr5GFP and Bmi1GFP-enriched populations with stem activity. Growth from singly isolated Lgr5GFP ISCs gave rise to small spheroids. Spheroids did not express Lgr5GFP and instead up-regulated Bmi1GFP expression. Conversely, Bmi1-derived spheroids initiated Lgr5GFP expression as crypt domains were established. These data showed the functional utility of murine mAbs in the isolation and investigation of Lgr5GFP and Bmi1GFP ISC-enriched populations. Ex vivo analyses showed hierarchical plasticity between different ISC-expressing states; specifically Lgr5GFP ISCs gave rise to Bmi1GFP cells, and vice versa. These data highlight the impact of temporal and physiological context on unappreciated interactions between Lgr5GFP and Bmi1GFP cells during crypt formation.

Pediatric high grade gliomas (pHGG) are a highly lethal subgroup of brain tumors that often recur despite lack of measurable disease on imaging, indicating persistent undetected treatment-resistant disease. Measures of disease in the central nervous system tumors are hampered by their sensitive neuroanatomical location, the risk of serial biopsy and limitations in imaging techniques. The development of liquid biomarkers for pHGG holds promise as a clinically relevant tool in diagnosis, prognosis, therapeutic predication, and disease surveillance. This review discusses the current state of liquid biopsy for pHGGs and its potential applications for biomarker development in pHGG.

Background/Objectives: Pancreatic ductal adenocarcinoma (PDAC) presents significant diagnostic and prognostic challenges, as current biomarkers frequently fail to accurately stage disease, predict rapid metastatic recurrence (rPDAC), or assess response to neoadjuvant therapy (NAT). We investigated the potential for circulating neoplastic-immune hybrid cells (CHCs) as a non-invasive, multifunctional biomarker for PDAC. Methods: Peripheral blood specimens were obtained from patients diagnosed with PDAC. CHCs were detected by co-expression of pan-cytokeratin and CD45, normalized to 50,000 peripheral blood mononuclear cells. rPDAC was defined as metastatic recurrence within six months of margin-negative pancreatectomy. Cyclic immunofluorescence (CyCIF) analyses compared hybrid phenotypes in blood and tumors. Results: Blood samples were collected from 42 patients with PDAC prior to resection. Those with radiographically occult metastatic disease and rPDAC had higher preoperative CHC numbers compared to patients who did not (65.0 and 74.4, vs. 11.52 CHCs; p < 0.001). Patients with complete or near-complete pathologic responses to NAT had lower preoperative CHC numbers than partial and/or non-responders (1.7 vs. 13.1 CHCs; p = 0.008). When assessed longitudinally, those with partial pathologic response saw CHC levels become undetectable while on treatment but increase in the interval between NAT completion and resection. In contrast, patients with poor responses or development of metastatic disease experienced persistent CHC detection during therapy or rising levels prior to radiographic evidence of metastases. Further, in metastatic PDAC patients, treatment-induced phenotypic changes in hybrid cells mirrored those in paired metastatic tumor samples. Conclusions: CHC enumeration and phenotyping display promise as a real-time indicator of disease burden, recurrence risk, and treatment response in PDAC. CHCs have great potential as tumor-derived biomarkers to optimize therapeutic strategies and improve survival in patients with PDAC.