I. INTRODUCTION

Section:

ChooseTop of pageABSTRACTI. INTRODUCTION <<II. MATERIALS AND METHODSIII. RESULTSIV. DISCUSSIONV. CONCLUSIONSSUPPLEMENTARY MATERIALREFERENCESNeoadjuvant chemotherapy (NAC) is used in breast cancer to reduce the need for mastectomies by shrinking the primary tumor.11. A. M. Thompson and S. L. Moulder-Thompson, “Neoadjuvant treatment of breast cancer,” Ann. Oncol. 23(Suppl 10), x231–x236 (2012). https://doi.org/10.1093/annonc/mds324,22. S. Masood, “Neoadjuvant chemotherapy in breast cancers,” Womens Health (London) 12, 480–491 (2016). https://doi.org/10.1177/1745505716677139 However, resistance to treatment can cause disease progression and promote distant organ metastasis.33. T. Ikeda, H. Jinno, A. Matsui, S. Masamura, and M. Kitajima, “The role of neoadjuvant chemotherapy for breast cancer treatment,” Breast Cancer 9, 8–14 (2002). https://doi.org/10.1007/BF02967540,44. A. Hanna, R. Birla, C. Iosif, M. Boeriu, and S. Constantinoiu, “Benefits and disadvantages of neoadjuvant radiochemotherapy (RCT) in the multimodal therapy of squamous esophageal cancer (ESC),” Chirurgia (Bucur) 111, 12–25 (2016). Therefore, longitudinal monitoring of NAC response is needed to identify resistant tumors early so that second-line therapies can be appropriately instituted. Currently, the response to chemotherapy is clinically determined by calculating the residual tumor burden at the time of surgery after completion of proposed chemotherapy. Clearly, identifying tumor resistance before completing the entire regimen of NAC would avoid unnecessary doses of toxic treatment, and predicting a complete pathological response may preclude the need for surgery. However, longitudinal monitoring using serial tissue biopsies is impractical. In contrast, routine blood draws are a standard practice in the clinic, making liquid biopsy an attractive approach to address these dilemmas. Thus, identifying blood-based biomarkers in breast cancer patients that can predict NAC response is essential for personalized cancer treatment.Liquid biopsy is an effective approach to track circulating tumor cells (CTCs) in metastatic breast cancer patients.5–75. S. F. Moussavi-Harami, K. B. Wisinski, and D. J. Beebe, “Circulating tumor cells in metastatic breast cancer: A prognostic and predictive marker,” J. Patient Cent. Res. Rev. 1, 85–92 (2014). https://doi.org/10.17294/2330-0698.10176. A. Giordano and M. Cristofanilli, “CTCs in metastatic breast cancer,” Recent Results Cancer Res. 195, 193–201 (2012). https://doi.org/10.1007/978-3-642-28160-0_187. S. K. Arya, B. Lim, and A. R. A. Rahman, “Enrichment, detection and clinical significance of circulating tumor cells,” Lab Chip 13, 1995–2027 (2013). https://doi.org/10.1039/c3lc00009e CTCs have been identified as critical determinants of the onset and advancement of metastasis.8–108. P. Rodrigues and S. Vanharanta, “Circulating tumor cells: Come together, right Now, over metastasis,” Cancer Discov. 9, 22–24 (2019). https://doi.org/10.1158/2159-8290.CD-18-12859. D. S. Micalizzi, S. Maheswaran, and D. A. Haber, “A conduit to metastasis: Circulating tumor cell biology,” Genes Dev. 31, 1827–1840 (2017). https://doi.org/10.1101/gad.305805.11710. C. Alix-Panabières and K. Pantel, “Technologies for detection of circulating tumor cells: Facts and vision,” Lab Chip 14, 57–62 (2014). https://doi.org/10.1039/C3LC50644D CTCs are the primary tumor cells that exit the breast parenchyma, enter the bloodstream, and survive systemic circulation. CTCs further rewire themselves and grow in distant organs, causing the spread of cancer. Since mortality in breast cancer patients undergoing neoadjuvant chemotherapy results from disease progression caused by metastasis,1111. A. J. Redig and S. S. McAllister, “Breast cancer as a systemic disease: A view of metastasis,” J. Intern. Med. 274, 113–126 (2013). https://doi.org/10.1111/joim.12084,1212. C. Selli and A. H. Sims, “Neoadjuvant therapy for breast cancer as a model for translational research,” Breast Cancer: Basic Clin. Res. 13, 117822341982907–1178223419829072 (2019). https://doi.org/10.1177/1178223419829072 technologies evaluating CTCs in the blood of patients undergoing NAC are needed.Breast cancer also initiates a significant host immune response, invoking inflammatory cells such as natural killer cells, T-cells, macrophages, etc.1313. H. Gonzalez, C. Hagerling, and Z. Werb, “Roles of the immune system in cancer: From tumor initiation to metastatic progression,” Genes Dev. 32, 1267–1284 (2018). https://doi.org/10.1101/gad.314617.118,1414. L. J. Standish et al., “Breast cancer and the immune system,” J. Soc. Integr. Oncol. 6, 158–168 (2008). Intriguingly, recent studies show cancer-associated macrophage-like cells (CAMLs) in the blood of breast, prostate, pancreatic, esophageal, lung, and renal cell cancer patients.15–2015. D. L. Adams et al., “Circulating giant macrophages as a potential biomarker of solid tumors,” Proc. Natl. Acad. Sci. U.S.A. 111, 3514–3519 (2014). https://doi.org/10.1073/pnas.132019811116. D. L. Adams et al., “Circulating cancer-associated macrophage-like cells differentiate malignant breast cancer and benign breast conditions,” Cancer Epidemiol. Biomarkers Prev. 25, 1037–1042 (2016). https://doi.org/10.1158/1055-9965.EPI-15-122117. D. J. Gironda et al., “Cancer associated macrophage-like cells and prognosis of esophageal cancer after chemoradiation therapy,” J. Transl. Med. 18, 413 (2020). https://doi.org/10.1186/s12967-020-02563-x18. P. Zhu et al., “Detection of tumor-associated cells in cryopreserved peripheral blood mononuclear cell samples for retrospective analysis,” J. Transl. Med. 14, 198 (2016). https://doi.org/10.1186/s12967-016-0953-219. Z. Mu et al., “Prognostic values of cancer associated macrophage-like cells (CAML) enumeration in metastatic breast cancer,” Breast Cancer Res. Treat. 165, 733–741 (2017). https://doi.org/10.1007/s10549-017-4372-820. A. Augustyn et al., “Giant circulating cancer-associated macrophage-like cells are associated with disease recurrence and survival in non-small-cell lung cancer treated with chemoradiation and atezolizumab,” Clin. Lung Cancer 22, e451–e465 (2021). https://doi.org/10.1016/j.cllc.2020.06.016 These reports suggest that CAMLs are either involved in engulfing tumor cells or aiding the transport of primary tumor cells during circulation.15–2115. D. L. Adams et al., “Circulating giant macrophages as a potential biomarker of solid tumors,” Proc. Natl. Acad. Sci. U.S.A. 111, 3514–3519 (2014). https://doi.org/10.1073/pnas.132019811121. M. T. Quinn and I. A. Schepetkin, “Role of NADPH oxidase in formation and function of multinucleated giant cells,” J. Innate Immun. 1, 509–526 (2009). https://doi.org/10.1159/000228158 As CAMLs and CTCs are both present in the blood of cancer patients15–2215. D. L. Adams et al., “Circulating giant macrophages as a potential biomarker of solid tumors,” Proc. Natl. Acad. Sci. U.S.A. 111, 3514–3519 (2014). https://doi.org/10.1073/pnas.132019811122. M. G. Krebs, J.-M. Hou, T. H. Ward, F. H. Blackhall, and C. Dive, “Circulating tumour cells: Their utility in cancer management and predicting outcomes,” Ther. Adv. Med. Oncol. 2, 351–365 (2010). https://doi.org/10.1177/1758834010378414 and represent the delicate balance between the oncogenic spread and host immune response, it is desirable to develop methods that enable simultaneous investigation of these cells. Such techniques will enable comprehensive profiling of rare circulating cells indicative of metastatic potential (CTCs) and host immunity (CAMLs), which can be a compelling means to predict NAC response in patients using routine blood draws.Prior approaches to isolate CTCs from the blood of breast cancer patients undergoing NAC have used antibody-based markers.23–2623. C. Hall et al., “Circulating tumor cells after neoadjuvant chemotherapy in stage I–III triple-negative breast cancer,” Ann. Surg. Oncol. 22, 552–558 (2015). https://doi.org/10.1245/s10434-015-4600-624. W. Onstenk et al., “Improved circulating tumor cell detection by a combined EpCAM and MCAM CellSearch enrichment approach in patients with breast cancer undergoing neoadjuvant chemotherapy,” Mol. Cancer Ther. 14, 821 (2015). https://doi.org/10.1158/1535-7163.MCT-14-065325. M. J. Serrano et al., “Dynamics of circulating tumor cells in early breast cancer under neoadjuvant therapy,” Exp. Ther. Med. 4, 43–48 (2012). https://doi.org/10.3892/etm.2012.54026. S. Kasimir-Bauer et al., “Does primary neoadjuvant systemic therapy eradicate minimal residual disease? Analysis of disseminated and circulating tumor cells before and after therapy,” Breast Cancer Res. 18, 20 (2016). https://doi.org/10.1186/s13058-016-0679-3 As shown in Table I, these affinity-based technologies use epithelial (E+) markers such as EpCAM (epithelial cellular adhesion molecule) and cytokeratin to isolate specific groups of CTCs. Given that tumor progression involves epithelial to mesenchymal transition and host immune response, capture and enumeration of only E+ CTCs remain a significant limitation of affinity-based techniques27–2927. A. Kowalik, M. Kowalewska, and S. Góźdź, “Current approaches for avoiding the limitations of circulating tumor cells detection methods—Implications for diagnosis and treatment of patients with solid tumors,” Transl. Res. 185, 58–84.e15 (2017). https://doi.org/10.1016/j.trsl.2017.04.00228. K. Pantel and M. R. Speicher, “The biology of circulating tumor cells,” Oncogene 35, 1216–1224 (2016). https://doi.org/10.1038/onc.2015.19229. Z. Shen, A. Wu, and X. Chen, “Current detection technologies for circulating tumor cells,” Chem. Soc. Rev. 46, 2038–2056 (2017). https://doi.org/10.1039/C6CS00803H as shown in Table I. Indeed, studies show that CTCs also exhibit mesenchymal (M+ CTCs) markers such as vimentin, N-cadherin, and fibronectin and can stain positive for both E+ and M+ markers (E+M+ CTCs).3030. R. Kalluri and R. A. Weinberg, “The basics of epithelial-mesenchymal transition,” J. Clin. Invest. 119, 1420–1428 (2009). https://doi.org/10.1172/JCI39104,3131. S. Lamouille, J. Xu, and R. Derynck, “Molecular mechanisms of epithelial-mesenchymal transition,” Nat. Rev. Mol. Cell Biol. 15, 178–196 (2014). https://doi.org/10.1038/nrm3758 It is crucial to enumerate the mesenchymal-type CTCs since they are known to be responsible for chemoresistance.3232. X.-X. Jie, X.-Y. Zhang, and C.-J. Xu, “Epithelial-to-mesenchymal transition, circulating tumor cells and cancer metastasis: Mechanisms and clinical applications,” Oncotarget 8, 81558–81571 (2017). https://doi.org/10.18632/oncotarget.18277,3333. T. Brabletz, R. Kalluri, M. A. Nieto, and R. A. Weinberg, “EMT in cancer,” Nat. Rev. Cancer 18, 128–134 (2018). https://doi.org/10.1038/nrc.2017.118 Thus, marker-based isolation technologies do not allow comprehensive profiling of CTCs and CAMLs in the blood of NAC patients.

TABLE I. CTC studies in breast cancer patients undergoing neoadjuvant chemotherapy and the technologies used for isolating rare cells in their blood.

StudyTechnologyRare cells enumeratedMarker-based Rare Cell Isolation technologiesSerrano et al.2525. M. J. Serrano et al., “Dynamics of circulating tumor cells in early breast cancer under neoadjuvant therapy,” Exp. Ther. Med. 4, 43–48 (2012). https://doi.org/10.3892/etm.2012.540Cytokeratin immunomagnetic cell separationE+ CTCsHall et al.2323. C. Hall et al., “Circulating tumor cells after neoadjuvant chemotherapy in stage I–III triple-negative breast cancer,” Ann. Surg. Oncol. 22, 552–558 (2015). https://doi.org/10.1245/s10434-015-4600-6Cell searchE+ CTCsOnstenk et al.2020. A. Augustyn et al., “Giant circulating cancer-associated macrophage-like cells are associated with disease recurrence and survival in non-small-cell lung cancer treated with chemoradiation and atezolizumab,” Clin. Lung Cancer 22, e451–e465 (2021). https://doi.org/10.1016/j.cllc.2020.06.016Cell searchE+ CTCsBauer et al.2626. S. Kasimir-Bauer et al., “Does primary neoadjuvant systemic therapy eradicate minimal residual disease? Analysis of disseminated and circulating tumor cells before and after therapy,” Breast Cancer Res. 18, 20 (2016). https://doi.org/10.1186/s13058-016-0679-3AdnaTestIsolated cells were not enumeratedPierga et al.4040. J. Y. Pierga et al., “Circulating tumour cells and pathological complete response: Independent prognostic factors in inflammatory breast cancer in a pooled analysis of two multicentre phase II trials (BEVERLY-1 and -2) of neoadjuvant chemotherapy combined with bevacizumab,” Ann. Oncol. 28, 103–109 (2017). https://doi.org/10.1093/annonc/mdw535Cell searchE+ CTCsRiethdorf et al.4141. S. Riethdorf et al., “Prognostic impact of circulating tumor cells for breast cancer patients treated in the neoadjuvant ‘geparquattro’ trial,” Clin. Cancer Res. 23, 5384–5393 (2017). https://doi.org/10.1158/1078-0432.CCR-17-0255Cell searchE+ CTCsMarker-free Rare Cell Isolation technologiesGwark et al., 20203434. S. Gwark et al., “Analysis of the serial circulating tumor cell count during neoadjuvant chemotherapy in breast cancer patients,” Sci. Rep. 10, 17466 (2020). https://doi.org/10.1038/s41598-020-74577-wSmart biopsy system isolation kitE+ CTCsNi et al.7–357. S. K. Arya, B. Lim, and A. R. A. Rahman, “Enrichment, detection and clinical significance of circulating tumor cells,” Lab Chip 13, 1995–2027 (2013). https://doi.org/10.1039/c3lc00009e35. C. Ni et al., “Prospective study of the relevance of circulating tumor cell status and neoadjuvant chemotherapy effectiveness in early breast cancer,” Cancer Med. 9, 2290–2298 (2020). https://doi.org/10.1002/cam4.2876CanPatrolIsolated cells were not enumeratedJakabova et al.3636. A. Jakabova et al., “Characterization of circulating tumor cells in early breast cancer patients receiving neoadjuvant chemotherapy,” Ther. Adv. Med. Oncol. 13, 175883592110284 (2021). https://doi.org/10.1177/17588359211028492MetaCellIsolated cells were not enumeratedThis studyLabyrinthE+ CTCs, M+ CTCs, E+M+ CTCs and CAMLsAddressing the limitation of marker-based technologies, label-free or antibody-independent technologies have been used to isolate CTCs in NAC patients.34–3634. S. Gwark et al., “Analysis of the serial circulating tumor cell count during neoadjuvant chemotherapy in breast cancer patients,” Sci. Rep. 10, 17466 (2020). https://doi.org/10.1038/s41598-020-74577-w35. C. Ni et al., “Prospective study of the relevance of circulating tumor cell status and neoadjuvant chemotherapy effectiveness in early breast cancer,” Cancer Med. 9, 2290–2298 (2020). https://doi.org/10.1002/cam4.287636. A. Jakabova et al., “Characterization of circulating tumor cells in early breast cancer patients receiving neoadjuvant chemotherapy,” Ther. Adv. Med. Oncol. 13, 175883592110284 (2021). https://doi.org/10.1177/17588359211028492 For example, Gwark et al.3434. S. Gwark et al., “Analysis of the serial circulating tumor cell count during neoadjuvant chemotherapy in breast cancer patients,” Sci. Rep. 10, 17466 (2020). https://doi.org/10.1038/s41598-020-74577-w used the Smart Biopsy System Isolation kit3737. S. J. Lee et al., “Evaluation of a novel approach to circulating tumor cell isolation for cancer gene panel analysis in patients with breast cancer,” Oncol. Lett. 13, 3025–3031 (2017). https://doi.org/10.3892/ol.2017.5807 to isolate CTCs from the blood of patients undergoing NAC. However, the study did not enumerate the mesenchymal phenotypes of the CTCs and CAMLs. Ni et al.3535. C. Ni et al., “Prospective study of the relevance of circulating tumor cell status and neoadjuvant chemotherapy effectiveness in early breast cancer,” Cancer Med. 9, 2290–2298 (2020). https://doi.org/10.1002/cam4.2876 used CanPatrol™3838. S. Wu et al., “Classification of circulating tumor cells by epithelial-mesenchymal transition markers,” PLos One 10, e0123976 (2015). https://doi.org/10.1371/journal.pone.0123976 technology and ribonucleic acid-in situ hybridization (RNA-ISH) to identify the expression of epithelial and mesenchymal genes in isolated cells, which enabled the classification of patients as CTC-positive and CTC-negative. Similarly, Jakabova et al.3636. A. Jakabova et al., “Characterization of circulating tumor cells in early breast cancer patients receiving neoadjuvant chemotherapy,” Ther. Adv. Med. Oncol. 13, 175883592110284 (2021). https://doi.org/10.1177/17588359211028492 used the MetaCell3939. K. Kolostova, Y. Zhang, R. M. Hoffman, and V. Bobek, “In vitro culture and characterization of human lung cancer circulating tumor cells isolated by size exclusion from an orthotopic nude-mouse model expressing fluorescent protein,” J. Fluoresc. 24, 1531–1536 (2014). https://doi.org/10.1007/s10895-014-1439-3 size-based filtration technique to isolate CTCs and classify patients as CTC-positive and CTC-negative based on the quantitative polymerase chain reaction (qPCR) analysis. In summary, current studies have not interrogated the ability of marker-free technologies to comprehensively enumerate the various rare cells that could be found at different stages in patients selected for NAC.The focus of our investigation is to determine whether marker-free technology Labyrinth can be used to isolate and enumerate CTC phenotypes (E+, M+, and E+M+ CTCs) and CAMLs in patients selected for NAC. Labyrinth technology uses inertial focusing and isolates CTCs based on size and deformability.4242. E. Lin et al., “High-throughput microfluidic labyrinth for the label-free isolation of circulating tumor cells,” Cell Syst. 5, 295–304.e4 (2017). https://doi.org/10.1016/j.cels.2017.08.012 The long spiral channels and sharp turns of the Labyrinth microfluidic device help in the distinctive and efficient focusing of CTCs and blood cells. The basic mechanism for particle separation in curved channels involves the inertial lift force that stabilizes particle position (i.e., particle focusing), while the Dean drag force aids in lateral migration due to cross-sectional circulation (i.e., particle separation).42–4542. E. Lin et al., “High-throughput microfluidic labyrinth for the label-free isolation of circulating tumor cells,” Cell Syst. 5, 295–304.e4 (2017). https://doi.org/10.1016/j.cels.2017.08.01243. D. R. Gossett and D. D. Carlo, “Particle focusing mechanisms in curving confined flows,” Anal. Chem. 81, 8459–8465 (2009). https://doi.org/10.1021/ac901306y44. D. Di Carlo, D. Irimia, R. G. Tompkins, and M. Toner, “Continuous inertial focusing, ordering, and separation of particles in microchannels,” Proc. Natl. Acad. Sci. U.S.A. 104, 18892–18897 (2007). https://doi.org/10.1073/pnas.070495810445. A. Gangadhar and S. A. Vanapalli, “Inertial focusing of particles and cells in the microfluidic labyrinth device: Role of sharp turns,” Biomicrofluidics 16, 044114 (2022). https://doi.org/10.1063/5.0101582 In the Labyrinth device, the turns help to have long channels in a small footprint as well as tight curvatures, both of which increase the opportunity to focus smaller particles. Previously, the Labyrinth technology was shown to isolate heterogeneous CTCs and CTC clusters in metastatic breast, lung, pancreatic, and prostate cancer patients.42–4742. E. Lin et al., “High-throughput microfluidic labyrinth for the label-free isolation of circulating tumor cells,” Cell Syst. 5, 295–304.e4 (2017). https://doi.org/10.1016/j.cels.2017.08.01246. M. Zeinali et al., “High-throughput label-free isolation of heterogeneous circulating tumor cells and CTC clusters from non-small-cell lung cancer patients,” Cancers 12, 127 (2020). https://doi.org/10.3390/cancers1201012747. L. Rivera-Báez et al., “Expansion of circulating tumor cells from patients with locally advanced pancreatic cancer enable patient derived xenografts and functional studies for personalized medicine,” Cancers 12, 1011 (2020). https://doi.org/10.3390/cancers12041011 Still, it is unclear whether this technology has the capability to enumerate the low CTC counts typically associated with treatment-naïve non-metastatic breast cancer patients. In addition, the ability of Labyrinth to identify and enumerate CAMLs remains to be explored. This evaluation is necessary to inform on whether the baseline counts or the real-time change in those counts can be used to predict the treatment response to the NAC.

II. MATERIALS AND METHODS

Section:

ChooseTop of pageABSTRACTI. INTRODUCTIONII. MATERIALS AND METHODS <<III. RESULTSIV. DISCUSSIONV. CONCLUSIONSSUPPLEMENTARY MATERIALREFERENCESOur workflow for isolating rare cells from the blood of patients selected for NAC is shown in Fig. 1. The Labyrinth device is used to isolate specific cell populations based on size and deformability using the principle of inertial focusing.4242. E. Lin et al., “High-throughput microfluidic labyrinth for the label-free isolation of circulating tumor cells,” Cell Syst. 5, 295–304.e4 (2017). https://doi.org/10.1016/j.cels.2017.08.012 The total channel length of 637 mm is compressed in 11 long loops. The height of the channel is 100 μm and the width is 500 μm. The device has four outlets, where WBCs are collected in the first outlet, CTCs are collected in the second outlet, and other components (OC) of blood are collected in the third and fourth outlets. Immunostaining is subsequently performed to identify the different cell types. Below, we describe the detailed methodology associated with this workflow.

A. Blood collection

To study the baseline, we recruited treatment naïve non-metastatic breast cancer patients who consented to receive NAC at the UMC Cancer Center, Texas Tech University Health Sciences Center (TTUHSC). This study was approved by the Institutional Review Board (IRB, Protocol No. L19-043) of TTUHSC, Lubbock. Blood samples were collected from 21 non-metastatic treatment naïve patients undergoing NAC after obtaining written informed consent. Blood samples were collected in BD Vacutainer blood collection tubes (Franklin Lakes, NJ).

B. Depletion of red blood cells

Depletion of red blood cells (RBCs) from 5 ml of whole blood was done using Ficoll-Paque™ Plus (Cytiva Life Sciences, Marlborough, MA) density gradient.4848. I. J. Fuss, M. E. Kanof, P. D. Smith, and H. Zola, “Isolation of whole mononuclear cells from peripheral blood and cord blood,” Curr. Protoc. Immunol. 85, 7.1.1–7.1.8 (2009). https://doi.org/10.1002/0471142735.im0701s85 Ficoll-Paque™ was layered in 15 ml conical centrifuge tubes with 1:1 diluted blood in phosphate buffer saline (PBS, Gibco, Gaithersburg, MD), as per the manufacturer's protocol. After centrifugation, the buffy layer containing peripheral blood mononuclear cells was collected and diluted 5× with PBS to make a total volume of 25 ml for further processing through the Labyrinth chip.

C. CTC isolation

Labyrinth microfluidic chip was primed with 1% pluronic solution (Sigma Aldrich, St. Louis, MO) by flowing 1 ml of the solution at a flow rate of 100 μl/min, followed by 10 min of incubation to prevent cell adhesion to channel walls. After the pluronic treatment, the 5× diluted blood was run through the Labyrinth chip at 2.5 ml/min. The flow was allowed to stabilize for a minute, after which the product from the CTC outlet was collected (see Fig. 1). Images showing separation of cancer cells in the CTC outlet are shown in Fig. S1 in the supplementary material.

D. Immunostaining for cell enumeration

Isolated CTCs underwent the slide centrifugation process using Cytospin™ (Epredia, Kalamazoo, MI). The product collected from the CTC outlet was loaded into EZ Megafunnel™ (Epredia, Kalamazoo, MI), after which Cytospin™ coated cells in a single layer on poly-lysine-coated glass slides. The Cytospin was run at 800 rpm for 10 min. Furthermore, the cells were fixed using 4% paraformaldehyde (PFA, Thermo Fisher Scientific, Waltham, MA) for 10 min and then permeabilizations by 0.2% Triton X-100 (Sigma Aldrich, St. Louis, MO) for 3 min. After the permeabilization step, the slide was washed 3× with PBS for 5 min each. Blocking was done at room temperature using 10% normal goat serum (Thermo Fisher Scientific, Waltham, MA) for 30 min. A cocktail of primary antibodies was added to the slides made using mouse anti-human PanCK IgG1 (Bio-Rad, Hercules, CA), rabbit anti-human Vimentin (Abcam, Waltham, MA), and mouse anti-human CD45 IgG2 (Bio-Rad, Hercules, CA). The slides were incubated overnight with the antibody cocktail at 4 °C in a humidified chamber. After incubation, the slides were washed 3× with PBS, followed by secondary antibody cocktail incubation for 90 min. The secondary antibody cocktail consisted of goat anti-mouse IgG1 AF 546, goat anti-rabbit AF 647, and goat anti-mouse IgG2 AF 488. All the secondary antibodies were procured from Thermo Fisher Scientific. After secondary antibody incubation, the slides were washed 3× with PBS and mounted with Prolong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific, Waltham, MA).

E. Imaging

After immunostaining, an Olympus IX81 microscope (Waltham, MA) and a Hamamatsu digital camera (ImagEM X2 EM-CCD, Bridgewater, NJ) were used for imaging. The microscope was equipped with a Thorlabs automated stage (Newton, NJ) and was controlled by software Slidebook 6.1 (3i Intelligent Imaging Innovations Inc., Denver, CO). The images were acquired at 20× magnification under DAPI, FITC, TRITC, and Cy5 fluorescent filters with exposure times between 20 and 100 ms. The imaging resolution was 0.8 micrometers per pixel. Images were analyzed using a Slidebook Reader.

F. Cell identification

Approximately 2500 images were acquired under each fluorescent filter for every patient sample. Images were analyzed manually using a Slidebook 6 Reader (3i Intelligent Imaging Innovations Inc., Denver, CO) to determine the cell counts. The images were analyzed by simultaneously switching between DAPI, FITC (CD45), TRITC (Cytokeratin), and CY5 (Vimentin) signals across each image for identification of CTC phenotypes and CAMLs.

Figure 2 shows the cellular profiling in the blood of treatment naïve breast cancer patients. CTCs were identified as epithelial CTCs (E+ CTCs) if they were positive for nucleus (DAPI) and cytokeratin (TRITC).46–5246. M. Zeinali et al., “High-throughput label-free isolation of heterogeneous circulating tumor cells and CTC clusters from non-small-cell lung cancer patients,” Cancers 12, 127 (2020). https://doi.org/10.3390/cancers1201012749. Y. Horimoto et al., “Analysis of circulating tumour cell and the epithelial mesenchymal transition (EMT) status during eribulin-based treatment in 22 patients with metastatic breast cancer: A pilot study,” J. Transl. Med. 16, 287 (2018). https://doi.org/10.1186/s12967-018-1663-850. S. Zhang et al., “Mesenchymal phenotype of circulating tumor cells is associated with distant metastasis in breast cancer patients,” Cancer Manag. Res. 9, 691–700 (2017). https://doi.org/10.2147/CMAR.S14980151. Z. Wang et al., “Perioperative circulating tumor cells (CTCs), MCTCs, and CTC-white blood cells detected by a size-based platform predict prognosis in renal cell carcinoma,” Dis. Markers 2021, 9956142. https://doi.org/10.1155/2021/995614252. S. L. Stott et al., “Isolation of circulating tumor cells using a microvortex-generating herringbone-chip,” Proc. Natl. Acad. Sci. U.S.A. 107, 18392 (2010). https://doi.org/10.1073/pnas.1012539107 Transitioning CTCs (E+M+ CTCs) were positive for nucleus (DAPI), cytokeratin (FITC), and vimentin (Cy5) markers.50–5350. S. Zhang et al., “Mesenchymal phenotype of circulating tumor cells is associated with distant metastasis in breast cancer patients,” Cancer Manag. Res. 9, 691–700 (2017). https://doi.org/10.2147/CMAR.S14980151. Z. Wang et al., “Perioperative circulating tumor cells (CTCs), MCTCs, and CTC-white blood cells detected by a size-based platform predict prognosis in renal cell carcinoma,” Dis. Markers 2021, 9956142. https://doi.org/10.1155/2021/995614253. W. Zhao et al., “Tumor antigen-independent and cell size variation-inclusive enrichment of viable circulating tumor cells,” Lab Chip 19, 1860–1876 (2019). https://doi.org/10.1039/C9LC00210C Similarly, CTCs were classified as mesenchymal if they were positive for nucleus (DAPI) and Vimentin (Cy5).49–5549. Y. Horimoto et al., “Analysis of circulating tumour cell and the epithelial mesenchymal transition (EMT) status during eribulin-based treatment in 22 patients with metastatic breast cancer: A pilot study,” J. Transl. Med. 16, 287 (2018). https://doi.org/10.1186/s12967-018-1663-850. S. Zhang et al., “Mesenchymal phenotype of circulating tumor cells is associated with distant metastasis in breast cancer patients,” Cancer Manag. Res. 9, 691–700 (2017). https://doi.org/10.2147/CMAR.S14980151. Z. Wang et al., “Perioperative circulating tumor cells (CTCs), MCTCs, and CTC-white blood cells detected by a size-based platform predict prognosis in renal cell carcinoma,” Dis. Markers 2021, 9956142. https://doi.org/10.1155/2021/995614254. G. Kallergi et al., “Epithelial to mesenchymal transition markers expressed in circulating tumour cells of early and metastatic breast cancer patients,” Breast Cancer Res. 13, R59 (2011). https://doi.org/10.1186/bcr289655. Y. Manjunath et al., “PD-L1 expression with epithelial mesenchymal transition of circulating tumor cells is associated with poor survival in curatively resected non-small cell lung cancer,” Cancers (Basel) 11, 806 (2019). https://doi.org/10.3390/cancers11060806 Manual scoring of different cell types did not present ambiguity except for some epithelial CTCs, in which case it was classified as an epithelial CTC only if the cytokeratin expression was 50% higher than a reference WBC.To identify cells as CAMLs, we used the same criteria as that of Adams et al. (for pancreatic, prostate, and breast cancers),15–2015. D. L. Adams et al., “Circulating giant macrophages as a potential biomarker of solid tumors,” Proc. Natl. Acad. Sci. U.S.A. 111, 3514–3519 (2014). https://doi.org/10.1073/pnas.132019811117. D. J. Gironda et al., “Cancer associated macrophage-like cells and prognosis of esophageal cancer after chemoradiation therapy,” J. Transl. Med. 18, 413 (2020). https://doi.org/10.1186/s12967-020-02563-x18. P. Zhu et al., “Detection of tumor-associated cells in cryopreserved peripheral blood mononuclear cell samples for retrospective analysis,” J. Transl. Med. 14, 198 (2016). https://doi.org/10.1186/s12967-016-0953-219. Z. Mu et al., “Prognostic values of cancer associated macrophage-like cells (CAML) enumeration in metastatic breast cancer,” Breast Cancer Res. Treat. 165, 733–741 (2017). https://doi.org/10.1007/s10549-017-4372-820. A. Augustyn et al., “Giant circulating cancer-associated macrophage-like cells are associated with disease recurrence and survival in non-small-cell lung cancer treated with chemoradiation and atezolizumab,” Clin. Lung Cancer 22, e451–e465 (2021). https://doi.org/10.1016/j.cllc.2020.06.016 Gironda et al. (for esophageal cancer),1717. D. J. Gironda et al., “Cancer associated macrophage-like cells and prognosis of esophageal cancer after chemoradiation therapy,” J. Transl. Med. 18, 413 (2020). https://doi.org/10.1186/s12967-020-02563-x Zhu et al. (renal cell carcinoma),1818. P. Zhu et al., “Detection of tumor-associated cells in cryopreserved peripheral blood mononuclear cell samples for retrospective analysis,” J. Transl. Med. 14, 198 (2016). https://doi.org/10.1186/s12967-016-0953-2 and Augustyn et al. (for lung cancer).2020. A. Augustyn et al., “Giant circulating cancer-associated macrophage-like cells are associated with disease recurrence and survival in non-small-cell lung cancer treated with chemoradiation and atezolizumab,” Clin. Lung Cancer 22, e451–e465 (2021). https://doi.org/10.1016/j.cllc.2020.06.016 In these studies, and our work, CAMLs were identified as cells exhibiting nucleus (DAPI), CD45 (FITC), and cytokeratin (TRITC).15–2015. D. L. Adams et al., “Circulating giant macrophages as a potential biomarker of solid tumors,” Proc. Natl. Acad. Sci. U.S.A. 111, 3514–3519 (2014). https://doi.org/10.1073/pnas.132019811117. D. J. Gironda et al., “Cancer associated macrophage-like cells and prognosis of esophageal cancer after chemoradiation therapy,” J. Transl. Med. 18, 413 (2020). https://doi.org/10.1186/s12967-020-02563-x18. P. Zhu et al., “Detection of tumor-associated cells in cryopreserved peripheral blood mononuclear cell samples for retrospective analysis,” J. Transl. Med. 14, 198 (2016). https://doi.org/10.1186/s12967-016-0953-219. Z. Mu et al., “Prognostic values of cancer associated macrophage-like cells (CAML) enumeration in metastatic breast cancer,” Breast Cancer Res. Treat. 165, 733–741 (2017). https://doi.org/10.1007/s10549-017-4372-820. A. Augustyn et al., “Giant circulating cancer-associated macrophage-like cells are associated with disease recurrence and survival in non-small-cell lung cancer treated with chemoradiation and atezolizumab,” Clin. Lung Cancer 22, e451–e465 (2021). https://doi.org/10.1016/j.cllc.2020.06.016 We also measured the size of CAMLs in representative samples and found it to range from 14 to 150 μm, which was congruent with the size range of 14–300 μm reported by Adams et al.15