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Purpose: There are currently no predictive molecular biomarkers to identify patients with oligometastatic disease (OMD) who will benefit from definitive-intent radiation therapy (RT). We prospectively characterized circulating tumor cell (CTC) kinetics in patients with OMD undergoing definitive-intent RT., Methods: This prospective correlative biomarker study included patients with any solid malignancy ≤5 metastatic sites in ≤3 anatomic organ systems undergoing definitive-intent RT to all disease sites. Circulating tumor cells (CTCs) were captured and enumerated using a biomimetic cell rolling and nanotechnology-based assay functionalized with antibodies against epithelial cell adhesion molecule, against human epidermal growth factor receptor 2, and against epidermal growth factor receptor before and during RT and at follow-up visits up to 2 years post-RT., Results: We enrolled 43 patients with a median follow-up of 14.3 months. The pretreatment CTC level (cells captured/mL) was not associated with the number of disease sites (median one metastatic site/patient, range 1-5) or metastasis location (bone, brain, visceral) on Wilcoxon signed-rank test, P > .05. Post-RT, 56% of patients received systemic therapy, and 72% of patients experienced subsequent local or systemic progression. For 90% of patients, a CTC level <15 within 130 days post-RT corresponded to a durable control of irradiated lesions. Patients with a favorable versus an unfavorable clearance profile experienced significantly longer progression-free survival after RT (median 13 v 4 months, log-rank test, P = .0011). On logistic regression, CTC level >15 at a given time point was associated with clinical disease progression within the subsequent 6 months (odds ratio 3.31, P = .007). In 26% of patients with disease progression, a CTC level >15 preceded radiographic or clinical progression., Conclusion: CTCs may serve as a biomarker for disease control in OMD and may predict disease progression before standard assessments for patients receiving diverse cancer-directed therapies.

Head and neck squamous cell carcinoma (HNSCC) is a common and deadly cancer. Circulating tumor cell (CTC) abundance may a valuable, prognostic biomarker in low- and intermediate-risk patients. However, few technologies have demonstrated success in detecting CTCs in these populations. We prospectively collected longitudinal CTC counts from two cohorts of patients receiving treatments at our institution using a highly sensitive device that purifies CTCs using biomimetic cell rolling and dendrimer-conjugated antibodies. In patients with intermediate risk human papillomavirus (HPV)-positive HNSCC, elevated CTC counts were detected in 13 of 14 subjects at screening with a median of 17 CTC/ml (range 0.2-2986.5). A second cohort of non-metastatic, HPV- HNSCC subjects received cetuximab monotherapy followed by surgical resection. In this cohort, all subjects had elevated baseline CTC counts median of 73 CTC/ml (range 5.4-332.9) with statistically significant declines during treatment. Interestingly, two patients with recurrent disease had elevated CTC counts during and following treatment, which also correlated with growth of size and ki67 expression in the primary tumor. The results suggest that our device may be a valuable tool for evaluating the success of less intensive treatment regimens., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: SH and AZW are co-founders of, and have significant financial interest in, Capio Biosciences. MJP and JB are associated with Capio Biosciences. Capio Biosciences is an early-stage biotechnology company working to commercialize the liquid biopsy approach described in this manuscript., (Copyright © 2023 Elsevier B.V. All rights reserved.)

A highly sensitive, circulating tumor cell (CTC)-based liquid biopsy was used to monitor gastrointestinal cancer patients during treatment to determine if CTC abundance was predictive of disease recurrence. The approach used a combination of biomimetic cell rolling on recombinant E-selectin and dendrimer-mediated multivalent immunocapture at the nanoscale to purify CTCs from peripheral blood mononuclear cells. Due to the exceptionally high numbers of CTCs captured, a machine learning algorithm approach was developed to efficiently and reliably quantify abundance of immunocytochemically-labeled cells. A convolutional neural network and logistic regression model achieved 82.9% true-positive identification of CTCs with a false positive rate below 0.1% on a validation set. The approach was then used to quantify CTC abundance in peripheral blood samples from 27 subjects before, during, and following treatments. Samples drawn from the patients either prior to receiving radiotherapy or early in chemotherapy had a median 50 CTC ml -1 whole blood (range 0.6-541.6). We found that the CTC counts drawn 3 months post treatment were predictive of disease progression (p = .045). This approach to quantifying CTC abundance may be a clinically impactful in the timely determination of gastrointestinal cancer progression or response to treatment., Competing Interests: Declaration of competing interest MJP, JB, SL, AZW, and SH are affiliated with Capio Biosciences where SH and AZW have significant equity shares., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Sensitive detection of circulating tumor cells (CTCs) from patients' peripheral blood facilitates on-demand monitoring of tumor progression. However, clinically significant capture of renal cell carcinoma CTCs (RCC-CTCs) remains elusive due to their heterogenous surface receptor expression. Herein, a novel capture platform is developed to detect RCC-CTCs through integration of dendrimer-mediated multivalent binding, a mixture of antibodies, and biomimetic cell rolling. The nanoscale binding kinetics measured using atomic force microscopy reveal that dendrimer-coated surfaces exhibit an order of magnitude enhancement in off-rate kinetics compared to surface without dendrimers, which translated into cell capture improvements by ~60%. Selectin-induced cell rolling facilitates surface recruitment of cancer cells, further improving cancer cell capture by up to 1.7-fold. Lastly, an antibody cocktail targeting four RCC-CTC surface receptors, which included epithelial cell adhesion molecule (EpCAM), carbonic anhydrase IX (CA9), epidermal growth factor receptor (EGFR), and hepatocyte growth factor receptor (c-Met), improves the capture of RCC cells by up to 80%. The optimal surface configuration outperforms the conventional assay solely relying on EpCAM, as demonstrated by detecting significantly more CTCs in patients' samples (9.8 ± 5.1 vs. 1.8 ± 2.0 CTCs mL -1 ). These results demonstrate that the newly engineered capture platform effectively detects RCC-CTCs for their potential use as tumor biomarkers., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: S. Hong and A.Z. Wang are co-founders of Capio Biosciences, Inc., a biotech startup that is commercializing CapioCyte™ CTC technology. M.J. Poellmann is an employee of Capio Biosciences.T. Zhang has received research funding (to Duke) from Abbvie, Acerta, Janssen, Merck, Merrimack, Novartis, OmniSeq, Pfizer, and PGDx; speaking fees from Exelixis, Genentech, Roche, Sanofi Aventis, and Genomic Health; and has consulted for AstraZeneca, Bristol-Myers Squibb, Foundation Medicine, Janssen, Pfizer, Pharmacyclics, Amgen, and Sanofi Aventis. Stock ownership/employment (spouse) from Capio Biosciences., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Detection of circulating tumor cells (CTCs) relying on their expression of epithelial cell markers, such as epithelial cell adhesion molecule (EpCAM), has been commonly used. However, this approach unlikely captures CTCs that have undergone the process of epithelial-mesenchymal transition (EMT). In this study, we have induced EMT of in vitro prostate (PCa) and breast cancer (BCa) cell lines by treatment of transforming growth factor β 1 (TGFβ 1 ), a pleiotropic cytokine with transition-regulating activities. We found that the TGFβ 1 -treated, post-EMT cells exhibited up to a 45% reduction in binding affinity to antibodies against EpCAM (aEpCAM). To overcome this limitation, we designed our capture platform that integrates a unique combination of biomimetic cell rolling, dendrimer-mediated multivalent binding, and antibody cocktails of aEpCAM/aEGFR/aHER-2. Our capture surfaces resulted in up to 98% capture efficiency of post-EMT cells from mixtures of TGFβ 1 -treated and untreated cancer cells spiked in culture media and human blood. In a clinical pilot study, our CTC device was also able to capture rare CTCs from PCa patients with significantly enhanced capture sensitivity and purity compared to the control surface with aEpCAM only, demonstrating its potential to provide a reliable detection solution for CTCs regardless of their EMT status.

Purpose: We aimed to examine the effects of multivalent binding and biomimetic cell rolling on the sensitivity and specificity of circulating tumor cell (CTC) capture. We also investigated the clinical significance of CTCs and their kinetic profiles in patients with cancer undergoing radiotherapy treatment. Experimental Design: Patients with histologically confirmed primary carcinoma undergoing radiotherapy, with or without chemotherapy, were eligible for enrollment. Peripheral blood was collected prospectively at up to five time points, including before radiotherapy, at the first week, mid-point and final week of treatment, as well as 4 to 12 weeks after completion of radiotherapy. CTC capture was accomplished using a nanotechnology-based assay (CapioCyte) functionalized with aEpCAM, aHER-2, and aEGFR. Results: CapioCyte was able to detect CTCs in all 24 cancer patients enrolled. Multivalent binding via poly(amidoamine) dendrimers further improved capture sensitivity. We also showed that cell rolling effect can improve CTC capture specificity (% of captured cells that are CK + /CD45 - /DAPI + ) up to 38%. Among the 18 patients with sequential CTC measurements, the median CTC decreased from 113 CTCs/mL before radiotherapy to 32 CTCs/mL at completion of radiotherapy ( P = 0.001). CTCs declined throughout radiotherapy in patients with complete clinical and/or radiographic response, in contrast with an elevation in CTCs at mid or post-radiotherapy in the two patients with known pathologic residual disease. Conclusions: Our study demonstrated that multivalent binding and cell rolling can improve the sensitivity and specificity of CTC capture compared with multivalent binding alone, allowing reliable monitoring of CTC changes during and after treatment. Clin Cancer Res; 24(11); 2539-47. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Enumeration of circulating tumor cells (CTCs) of small-cell lung cancer (SCLC) patients has been shown to predict the disease progress and long-term survival. Most CTC detection methods rely on epithelial surface markers, such as epithelial cell adhesion molecule (EpCAM). However, this marker in SCLC is reported to be often downregulated after a variety of phenotypic changes, which impairs the reliability of EpCAM-based CTC detections. In this regard, the development of an alternative CTC detection method involving different CTC surface markers is in demand. In this study, we evaluated, for the first time to our knowledge, the feasibility of detecting SCLC CTCs using a noncatalytic endosialidase (EndoN Trap, EndoNt). This noncatalytic enzyme was chosen due to its high affinity to polysialic acid (polySia), a cell-surface glycan, that is highly expressed by SCLC tissue. Furthermore, this enzyme-based system was integrated into our dendrimer-mediated CTC capture platform to further enhance the capture efficiency via multivalent binding. We found that the EndoNt-immobilized surfaces could specifically capture polySia-positive SCLC cells and the binding between SCLC cells and EndoNt surfaces was further stabilized by dendrimer-mediated multivalent binding. When compared to the EpCAM-based capture, EndoNt significantly improved the capture efficiency of polySia-positive SCLC cells under flow due to its higher binding affinity (lower dissociation rate constants). These findings suggest that this enzyme-based CTC capture strategy has the potential to be used as a superior alternative to the commonly used EpCAM-based methods, particularly for those types of cancer that overexpress polySia.

Circulating tumor cells (CTCs) are cells that have detached from the primary tumor and entered circulation with potential to initiate a site of metastasis. Currently, CTC detection using CellSearch is cleared by the Food and Drug Administration for monitoring metastatic breast, prostate, and colorectal cancers as a prognostic biomarker for progression-free and overall survival. Accumulating evidence suggests CTCs have similar prognostic value in other metastatic and non-metastatic settings. Current research efforts are focused on extending the utility of CTCs beyond a prognostic biomarker to help guide clinical decision-making. These include using CTCs as a screening tool for diagnosis, liquid biopsy for molecular profiling, predictive biomarker to specific therapies, and monitoring tool to assess response and guide changes to treatment. CTCs have unique advantages vs circulating tumor DNA in this endeavor. Indications for CTCs in daily practice will expand as isolation techniques improve and clinical studies validating their utility continue to grow., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Circulating tumor cells (CTCs) have received a great deal of scientific and clinical attention as a biomarker for diagnosis and prognosis of many types of cancer. Given their potential significance in clinics, a variety of detection methods, utilizing the recent advances in nanotechnology and microfluidics, have been introduced in an effort of achieving clinically significant detection of CTCs. However, effective detection and isolation of CTCs still remain a tremendous challenge due to their extreme rarity and phenotypic heterogeneity. Among many approaches that are currently under development, this review paper focuses on a unique, promising approach that takes advantages of naturally occurring processes achievable through application of nanotechnology to realize significant improvement in sensitivity and specificity of CTC capture. We provide an overview of successful outcome of this biomimetic CTC capture system in detection of tumor cells from in vitro, in vivo, and clinical pilot studies. We also emphasize the clinical impact of CTCs as biomarkers in cancer diagnosis and predictive prognosis, which provides a cost-effective, minimally invasive method that potentially replaces or supplements existing methods such as imaging technologies and solid tissue biopsy. In addition, their potential prognostic values as treatment guidelines and that ultimately help to realize personalized therapy are discussed., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Although circulating tumor cells (CTCs) in blood have been widely investigated as a potential biomarker for diagnosis and prognosis of metastatic cancer, their inherent rarity and heterogeneity bring tremendous challenges to develop a CTC detection method with clinically significant specificity and sensitivity. With advances in nanotechnology, a series of new methods that are highly promising have emerged to enable or enhance detection and separation of CTCs from blood. In this review, we systematically categorize nanomaterials, such as gold nanoparticles, magnetic nanoparticles, quantum dots, graphenes/graphene oxides, and dendrimers and stimuli-responsive polymers, used in the newly developed CTC detection methods. This will provide a comprehensive overview of recent advances in the CTC detection achieved through application of nanotechnology as well as the challenges that these existing technologies must overcome to be directly impactful on human health., (© 2015 Wiley Periodicals, Inc.)

The lack of an effective detection method for lung circulating tumor cells (CTCs) presents a substantial challenge to elucidate the value of CTCs as a diagnostic or prognostic indicator in lung cancer, particularly in nonsmall cell lung cancer (NSCLC). In this study, we prepared a capture surface exploiting strong multivalent binding mediated by poly(amidoamine) (PAMAM) dendrimers to capture CTCs originating from lung cancers. Given that 85% of the tumor cells from NSCLC patients overexpress epidermal growth factor receptor (EGFR), anti-EGFR was chosen as a capture agent. Following in vitro confirmation using the murine lung cancer cell lines (ED-1 and ED1-SC), cyclin E-overexpressing (CEO) transgenic mice were employed as an in vivo lung tumor model to assess specificity and sensitivity of the capture surface. The numbers of CTCs in blood from the CEO transgenic mice were significantly higher than those from the healthy controls (on average 75.3 ± 14.9 vs 4.4 ± 1.2 CTCs/100 μL of blood, p < 0.005), indicating the high sensitivity and specificity of our surface. Furthermore, we found that the capture surface also offers a simple, effective method for monitoring treatment responses, as observed by the significant decrease in the CTC numbers from the CEO mice upon a treatment using a novel anti-miR-31 locked nucleic acid (LNA), compared to a vehicle treatment and a control-LNA treatment (p < 0.05). This in vivo evaluation study confirms that our capture surface is highly efficient in detecting in vivo CTCs and thus has translational potential as a diagnostic and prognostic tool for lung cancer.

Here, we report a new method for multicomponent protein patterning in a microchannel and also a technique for improving immunoaffinity-based circulating tumor cell (CTC) capture by patterning regions of alternating adhesive proteins using the new method. The first of two proteins, antiepithelial cell adhesion molecule (anti-EpCAM), provides the specificity for CTC capture. The second, E-selectin, increases CTC capture under shear. Patterning regions with and without E-selectin allows captured leukocytes, which also bind E-selectin and are unwanted impurities in CTC isolation, to roll a short distance and detach from the capture surface. This reduces leukocyte capture by up to 82%. The patterning is combined with a leukocyte elution step in which a calcium chelating buffer effectively deactivates E-selectin so that leukocytes may be rinsed away 60% more efficiently than with a buffer containing calcium. The alternating patterning of this biomimetic protein combination, used in conjunction with the elution step, reduces capture of leukocytes while maintaining a high tumor cell capture efficiency that is up to 1.9 times higher than the tumor cell capture efficiency of a surface with only anti-EpCAM. The new patterning technique described here does not require mask alignment and can be used to spatially control the immobilization of any two proteins or protein mixtures inside a sealed microfluidic channel.

Many microfluidic devices operate with cells suspended in buffer solutions. Researchers who work with large cell types in such devices often run into problems with gravitational cell settling in the injection equipment and in the device itself. A method for reducing this problematic settling is discussed in this paper using tumor cell lines as an example. Microfluidic circulating tumor cell (CTC) isolation devices (MCIDs) are benchmarked using buffer solutions spiked with in-vitro tumor cell lines prior to validation with clinical samples (i.e. whole blood). However, buffer solutions have different rheological properties than whole blood. Here we describe the use of alginate in PBS buffer solutions to mimic blood rheology and reduce cell settling during preliminary validation experiments. Because alginate increases the viscosity of a solution, it helps to maintain cells in suspension. We report that vertical equipment configurations are important to further mitigate the effects of cell settling for MDA-MB-468 carcinoma cells. We also report that alginate does not disrupt the specific binding interactions that are the basis of carcinoma cell capture in MCIDs. These results indicate that vertical equipment configurations and the addition of alginates can be used to reduce cell settling in buffer based MCID testing and other applications involving large cells suspended in buffer solution.

The selective detection of circulating tumor cells (CTCs) is of significant clinical importance for the clinical diagnosis and prognosis of cancer metastasis. However, largely because of the extremely low number of CTCs (as low as 1 in 10(9) hematologic cells) in the blood of patients, effective detection and separation of the rare cells remain a tremendous challenge. Cell rolling is known to play a key role in physiological processes such as the recruitment of leukocytes to sites of inflammation and selectin-mediated CTC metastasis. Furthermore, because CTCs typically express the epithelial-cell adhesion molecule (EpCAM) on the surface whereas normal hematologic cells do not, substrates with immobilized antibody against EpCAM may specifically interact with CTCs. In this article, we created biomimetic surfaces functionalized with P- and E-selectin and anti-EpCAM that induce different responses in HL-60 (used as a model of leukocytes in this study) and MCF-7 (a model of CTCs) cells. HL-60 and MCF-7 cells showed different degrees of interaction with P-/E-selectin and anti-EpCAM at a shear stress of 0.32 dyn/cm(2). HL-60 cells exhibited rolling on P-selectin-immobilized substrates at a velocity of 2.26 +/- 0.28 microm/s whereas MCF-7 cells had no interaction with the surface. Both cell lines, however, had interactions with E-selectin, and the rolling velocity of MCF-7 cells (4.24 +/- 0.31 microm/s) was faster than that of HL-60 cells (2.12 +/- 0.15 microm/s). However, only MCF-7 cells interacted with anti-EpCAM-coated surfaces, forming stationary binding under flow. More importantly, the combination of the rolling (E-selectin) and stationary binding (anti-EpCAM) resulted in substantially enhanced separation capacity and capture efficiency (more than 3-fold enhancement), as compared to a surface functionalized solely with anti-EpCAM that has been commonly used for CTC capture. Our results indicate that cell-specific detection and separation may be achieved through mimicking the biological processes of combined dynamic cell rolling and stationary binding, which will likely lead to a CTC detection device with significantly enhanced specificity and sensitivity without a complex fabrication process.