Phenotyping of tumor-associated circulating cells for metastatic spread prediction

Abstract

Certain embodiments are directed to methods including isolating CTCs and determining nanomechanical properties of the isolated cells that are randomly shed from tumor. Moreover, CTCs uniquely co-isolate with macrophage-like cells bearing the markers of tumor-associated macrophages (TAMs). Presence of these immune cells was indicative of survival-promoting phenotype of "mechanical fitness" in CTCs.

Description

FJ ref. UTSK-P0571US / Client ref HSC-1697PHENOTYPING OF TUMOR-ASSOCIATED CIRCULATING CELLS FOR METASTATIC SPREAD PREDICTIONPRIORITY

[0001] This application claims priority to U.S. Provisional Patent serial number 63 / 727,172 filed December 2, 2024, which is incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

[0002] This invention was made with government support under PC 170821 awarded by the Department of Defense as well as U01 CA283749 and CA217297-01 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Circulating tumor cells (CTCs) are shed by aggressive epithelial-origin tumors and are found in the bloodstream of patients at the risk of metastasis or with already detected metastatic growth (Micalizzi et al., 2017). CTCs are as rare as one in a billion of blood cells, however due to their unique properties such as large size and epithelial surface markers they can be isolated by microfiltration or immunoaffinity capture from a “liquid biopsy”: few milliliters of a patient’s peripheral blood (Jin et al., 2019). Since the bloodstream is not a natural environment for epithelial- like CTCs, majority of them die by mechanical stress, apoptosis or anoikis, or due to attacks from immune cells (Katt et al., 2018). The surviving few CTCs progress with an epithelial (E) to- mesenchymal (M) transition (EMT), extravasate and start new tumor growth in distant tissues (Jin et al., 2019; Follain et al., 2020). Enumeration of CTCs is used as a general prognostic biomarker, with serious attempts to a more advanced precision medicine related analysis (Jin et al., 2019). Indeed, the enum eration- only approach centered on EpCAM (epithelial cell adhesion molecule) positive cells neglect cells with advanced EMT and may miss the opportunity to determine metastatic potential among heterogeneous CTCs (Bitting et al., 2014; Gorges et al., 2012).

[0004] Since successful CTCs need a specific set of adaptations to withstand conditions in the bloodstream, the Inventors turned their attention to CTC physical endurance. Mechanical challenges during intravasation, circulation, and extravasation point at the physical properties of CTCs as crucial factors influencing the cells’ capability to survive the hostile environment (StrokaFJ ref. UTSK-P0571US / Client ref HSC-1697 and Konstantopoulos, 2014). These physical properties are tightly connected to EMT that involves a massive remodeling of the cytoskeleton and membranes (Savagner, 2010) affecting cell softness and adhesion. CTCs express a wide spectrum of both epithelial and mesenchymal marker proteins and EMT traits are considered biomarkers of poor prognosis (Chen et al., 2013; Yang et al., 2019). However, plakoglobin implicated in survival -promoting CTC clustering is a component of epithelial, not mesenchymal cell junctions (Aceto et al., 2014). Indeed, the recent evidence suggests that epithelial-mesenchymal plasticity (EMP): the ability to adopt and traverse intermediate E / M states (Yang et al., 2020), is a crucial adaptive strategy for CTCs surviving in the bloodstream (Chen et al., 2013; Lecharpentier et al., 2011; Pastushenko et al., 2018). The mechanism and extent of EMP in tumor cells and CTCs as well as its clinical significance in metastatic potential are not fully understood (Williams et al., 2019; Liao et al., 2020).

[0005] There remains a need for additional methods to phenotype circulating cancer cells.SUMMARY

[0006] One solution to problems presented above is to isolate CTCs and determine nanomechanical properties of the isolated cells that are randomly shed from a tumor. Moreover, CTCs uniquely co-isolate with macrophage-like cells bearing the markers of tumor-associated macrophages (TAMs). The presence of these immune cells was indicative of survival -promoting phenotype of “mechanical fitness” in CTCs.

[0007] Aggressive tumors of epithelial origin shed cells that intravasate into the bloodstream, become circulating tumor cells (CTCs), and either die or seed metastases. The inventors have discovered that CTCs isolated from the blood of prostate cancer patients display distinct nanomechanical phenotypes characterized by high adhesion and high softness. These phenotypes reflect the cells’ endurance, invasiveness, and the patient’s sensitivity to androgen-deprivation therapy (ADT). Patient-derived CTCs are nanomechanically distinct from tumor cells randomly shed into urine or from biopsy material, with markedly higher adhesion being the most prominent biophysical difference. Moreover, CTCs uniquely co-isolate with macrophage-like cells expressing markers of tumor-associated macrophages (TAMs). The abundance of these immune cells strongly correlates with a survival-promoting “mechanical fitness” phenotype in CTCs - defined as high softness (low stiffness) and high adhesion as measured by atomic force microscopy (AFM).FJ ref. UTSK-P0571US / Client ref HSC-1697

[0008] The mechanical fitness of CTCs is driven by epithelial-mesenchymal plasticity (EMP) and is actively modulated by the interplay between fluid shear stress (FSS) in the circulation and direct physical interactions between CTCs and tumor-associated macrophages (TAMs or circulating MGs / Mcs).

[0009] The methods described herein can be used for modelling FSS to explore conditions encountered in circulation by CTCs and MOs / Mcs. So far, studies on the role of FSS and contacts with leukocytes focused on intravasation or extravasation, relevant to post-FSS seeding. Methods can be used to define “aggressive CTCs” that would be most relevant to determine a risk factor of metastasis. The term “aggressive / invasive” is used commonly but rarely in a rigorous way.

[0010] The methods can be applied to multiparameter mechanical phenotyping of patient- isolated and model CTCs. To the best of our knowledge, we are the only group employing Peak Force Quantitative Nanomechanical Mapping (PF-QNM) AFM to probe live, patient-isolated CTCs. PF-QNM Atomic Force Microscopy is an advanced technique in atomic force microscopy (AFM) used for characterizing the mechanical properties of materials at the nanoscale. Unlike traditional AFM techniques, PF-QNM measures the sample's mechanical properties by applying a series of small indentation forces at each pixel of the scanned area and detecting the resulting displacement. This method allows for high-resolution mapping of properties such as elasticity, stiffness, adhesion, and viscosity. By providing quantitative data on material properties with high spatial resolution, PF-QNM AFM is valuable for various fields including materials science, biology, and nanotechnology. Other AFM studies of patient samples use tumor biopsies or tumor tissue. The studies on model CTCs employ standard stationary cultures. Importantly, the published reports are limited to single parameters such as elasticity or adhesion, the latter often approximated in a non-quantitative manner. Multiparameter phenotyping is contemplated as the most productive and rigorous approach to cells’ stratification.

[0011] The methods described herein can be used for collective classification of patient- isolated and model CTCs based on their mechanical phenotypes, EMP and encounters with immune cells. The methods can be used for mechanical comparison of tumor cells and CTCs from the same patient: a unique opportunity for insight into origins of CTCs’ properties.

[0012] Aspects of the invention can be used to enumerate distinct classes of CTCs and copurifying immune cells as a potential biomarker, expanding the use of liquid biopsies. EnumerationFJ ref. UTSK-P0571US / Client ref HSC-1697 of epithelial-like CTCs is approved for clinical use; however, it neglects the diversity and interactions of CTCs and suffers from low accuracy.

[0013] Certain embodiments are directed to methods for treating a cancer patient having or suspected of having an epithelial derived cancer comprising: (i) isolating tumor associated cancer cells (TACC) from a blood sample; (ii) assessing stiffness and adhesion properties of circulating tumor cells using atomic force microscopy to determine metastatic potential of circulating cancer cells; and (iii) treating a patient determined to be pre-metastatic or early metastatic based on stiffness and adhesion properties of circulating tumor cells. In specific embodiments, the cancer patient can be a lung cancer patient or a prostate cancer patient.

[0014] Other embodiments of the invention are discussed throughout this application. Any embodiment discussed with respect to one aspect of the invention applies to other aspects of the invention as well and vice versa. Each embodiment described herein is understood to be applicable to all aspects of the invention. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions and kits of the invention can be used to achieve methods of the invention.

[0015] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0016] Throughout this application, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0017] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.”

[0018] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open- ended and do not exclude additional, unrecited elements or method steps.

[0019] As used herein, the transitional phrases “consists of’ and “consisting of’ exclude any element, step, or component not specified. For example, “consists of’ or “consisting of’ used in aFJ ref. UTSK-P0571US / Client ref HSC-1697 claim would limit the claim to the components, materials or steps specifically recited in the claim except for impurities ordinarily associated therewith (i.e., impurities within a given component). When the phrase “consists of’ or “consisting of’ appears in a clause of the body of a claim the phrase “consists of’ or “consisting of’ limits only the elements (or components or steps) set forth in that clause; other elements (or components) are not excluded from the claim as a whole.

[0020] As used herein, the transitional phrases “consists essentially of’ and “consisting essentially of’ are used to define a chemical composition and / or method that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel character! stic(s) of the claimed invention. The term “consisting essentially of’ occupies a middle ground between “comprising” and “consisting of’.

[0021] As used in this specification and the appended claims, the following terms and phrases shall have the meanings set forth below unless clearly indicated otherwise:

[0022] “Circulating tumor cells” or “CTCs” refer to viable cells of epithelial or partial epithelial-mesenchymal origin shed from a primary or metastatic tumor into the bloodstream and identifiable by expression of epithelial markers (e.g., EpCAM, cytokeratins 8 / 18 / 19) and / or absence of CD45.

[0023] “Tumor-associated macrophages” or “TAMs” refer to CD45+immune cells co-isolated with CTCs that express one or more macrophage-lineage or polarization markers including, but not limited to, CD68, CD80, CD 163, CD206, CD204, HLA-DR, CXCR4, Tie-2, MertK, and arginase- 1.

[0024] “Tumor-associated circulating cells” or “TACCs” refer to the complete population of large (>8 pm), viable cells isolated from patient blood, bone marrow, effusion, or other body fluid by size-exclusion or equivalent viability-preserving methods, comprising CTCs, TAMs, and occasional CTC-macrophage hybrids or other tumor-derived immune companions.

[0025] “Intermediate-phenotype TAMs” or “intermediate macrophages” refer to TAMs coexpressing both Ml-like (e.g., CD80) and M2 -like (e g., CD163, CD206) polarization markers.

[0026] “M2-like TAMs” refer to TAMs predominantly expressing CD 163, CD206, and / or other alternative-polarization markers.

[0027] “Mechanical fitness” or “mechanically fit CTCs” refer to CTCs exhibiting a combination of high adhesion (typically >200 pN, more preferably >300 pN) and low corticalFJ ref. UTSK-P0571US / Client ref HSC-1697 stiffness (Young’s modulus typically <5 kPa, more preferably <3 kPa) as measured by atomic force microscopy on live cells.

[0028] “High adhesion” refers to a mean adhesion force of greater than 200 pN, preferably greater than 300 pN, and more preferably greater than 500 pN, as determined by PeakForce QNM or equivalent AFM mode.

[0029] “Low stiffness” or “high softness” refers to a Young’s modulus of less than 5 kPa, preferably less than 3 kPa, and more preferably less than 2 kPa, as determined by PeakForce QNM or equivalent AFM mode.

[0030] “Elevated metastatic risk” refers to a patient classification indicating significantly higher probability of radiographic progression, early castration resistance (in prostate cancer), or early recurrence (in lung cancer) within 6-24 months compared to patients lacking the mechanical fitness / TAM signature.

[0031] “Atomic force microscopy” or “AFM” encompasses PeakForce Quantitative Nanomechanical Mapping (PF-QNM), force-volume, contact, jumping, and other modes capable of quantitatively measuring adhesion, stiffness, deformation, and / or viscoelastic properties of live cells.

[0032] “Live CTCs” or “unfixed CTCs” refer to CTCs that have not been subjected to chemical fixation (e.g., formaldehyde, methanol) prior to nanomechanical measurement, thereby preserving native biomechanical properties.

[0033] “Patient with prostate cancer” includes individuals with localized, locally advanced, biochemically recurrent, non-metastatic castration-resistant, or metastatic castration-resistant disease, whether treatment-naive or undergoing androgen deprivation therapy.

[0034] “Patient with lung cancer” includes individuals with non-small-cell lung cancer (NSCLC) or small-cell lung cancer (SCLC) at any stage, including post-curative-intent resection, adjuvant therapy, or advanced / metastatic settings.

[0035] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.FJ ref. UTSK-P0571US / Client ref HSC-1697DESCRIPTION OF THE DRAWINGS

[0036] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of the specification embodiments presented herein.

[0037] FIG. 1. Natural history of CTCs and associated immune cells in early circulation phase (schematic).

[0038] FIG. 2. Mechanical parameters acquired in single AFM scans of live CTCs (schematic).

[0039] FIG. 3A-3D. Urine prostate cells (UPCs) vs. blood-derived CTCs: distinct nanomechanical properties and immune companions (representative images and PCA).

[0040] FIG. 4A-4C. Mechanical phenotyping of 514 CTCs from 33 blood samples (23 prostate cancer patients) reveals four mechanical classes; classes 3 and 4 represent highest “mechanical fitness.”

[0041] FIG. 5A-5D. Correlations between nanomechanical parameters of CTCs and enumeration of intermediate and M2-like macrophages.

[0042] FIG. 6A-6B. Prostate cancer cell lines co-cultured with model macrophages shift mechanical properties toward patient-isolated CTC profiles.

[0043] FIG. 7A-7C. Increased abundance of mechanically fit cells after co-culture with macrophages in three prostate cancer cell lines.

[0044] FIG. 8A-8F. CyTOF analysis of DU145 cells co-cultured with polarized macrophages shows hybrid EMT and partial mesenchymal-to-epithelial reversion.

[0045] FIG. 9. Representative PCA comparing mechanically fit CTCs (patient 12) vs. less fit CTCs (patient 008).

[0046] FIG. 10A-10B. CTCs from prostate and lung cancer patients share similar high- adhesion mechanical phenotypes distinct from respective tumor-derived cells.

[0047] FIG. 11A-11C. Cultured prostate and lung cancer cell lines as valid mechanical models; response to EMT modulation.

[0048] FIG. 12. FSS-surviving model cells become more adhesive and slightly less soft (combined 1 hr + 3 hr data).

[0049] FIG. 13A-13B. Macrophage enumeration correlates with CTC mechanical fitness; elevated putative CTC-macrophage fusions in metastatic patients.FJ ref. UTSK-P0571US / Client ref HSC-1697

[0050] FIG. 14A-14B. Co-culture with macrophages induces adhesion / deformation increase and hybrid EMT in DU145 cells.

[0051] FIG. 15A-15B. Prior exposure to M2 macrophages protects DU145 cells from FSS- induced stiffening and enhances adhesion.

[0052] FIG. 16. Convergence of mechanically fittest CTC fraction and macrophage fraction across 33 blood samples.

[0053] FIG. 17. Schematic summary of mechanical phenotype shifts induced by FSS and macrophage contact.

[0054] FIG. 18A-18C. Microfluidic circulation simulation setup (ibidi system) and representative images of cells under vein-like FSS.

[0055] FIG. 19A-19C. Macrophage co-culture increases resilience of DU145 cells to prolonged FSS; time-dependent cluster formation.

[0056] FIG. 20A-20C. Short (15 min) vein-like FSS shifts cultured prostate cancer cells toward patient-CTC nanomechanical phenotypes; benign BPH-1 cells unresponsive.

[0057] FIG. 21A-21B. Principle of PeakForce QNM AFM on live cells (schematic).

[0058] FIG. 22. Representative workflow screenshots from Nanoscope software.

[0059] FIG. 23A-23B. Example PF-QNM maps of a live pancreatic CTC and immunofluorescence identification of TACCs / TMHs on filter.

[0060] FIG. 24A-24D. Comparative mechanical phenotyping of CTCs vs. UPCs, TMHs, and prognostic grouping of patients using combined mechanical + enumeration data.

[0061] FIG. 25A-25B. Unsupervised clustering of 30 ADT-initiating patients by CTC mechanical parameters + TACC enumeration perfectly separates short vs. long progression-free survival.

[0062] FIG. 26A-26D. Classification of individual CTCs into four mechanical classes (A-D) and patient grouping by dominant class frequency.

[0063] FIG. 27A-27B. Cox regression and Kaplan-Meier showing >6 high-adhesion (>1.66 nN) CTCs per 7.5 mL predicts early castration resistance (HR 2.5).

[0064] FIG. 28A-28C. Enhanced predictive model incorporating EMT-CTC and intermediatemacrophage enumeration (HR 1.031 and 1.042 per additional cell).

[0065] FIG. 29. PCA of 19 mCRPC patients before second-line therapy; responders and nonresponders form therapy-specific non-overlapping clusters.FJ ref. UTSK-P0571US / Client ref HSC-1697

[0066] FIG. 30A-30D. Biophysical comparison of two stage TA3 NSCLC patients before resection; higher CTC adhesion and intermediate macrophages in the patient who recurred within 1 year.

[0067] FIG. 31A-31B. PCA biplot of >700 CTCs / TMHs revealing three distinct mechanical adaptation strategies (erythrocyte-like, leukocyte / amoeboid, adhesive / clustering).

[0068] FIG. 32. Immunofluorescence of heterotypic CTC-macrophage clusters exclusively in patients with adhesive / clustering mechanical phenotype.

[0069] FIG. 33. Representative tumor-macrophage hybrid (TMH) expressing both epithelial and macrophage markers with combined high adhesion / high deformability.

[0070] FIG. 34. Stage-specific shift in dominant mechanical predictor (adhesion —> deformation) from castration-sensitive to polymetastatic disease.

[0071] FIG. 35. Kaplan-Meier of oligo-to-poly progression in 28 CRPC patients stratified by amoeboid-dominant phenotype / TMH abundance (HR 4.8).DESCRIPTION

[0072] The following description is directed to various embodiments of the invention. The term “invention” is not intended to refer to any particular embodiment or otherwise limit the scope of the disclosure. Although one or more of these embodiments may be preferred, the embodiments disclosed should not be interpreted as limiting the scope of the disclosure, including the claims. In addition, one skilled in the art will understand that the following description has broad application, and the discussion of any embodiment is meant only to be an example of that embodiment and not intended to imply that the scope of the disclosure, including the claims, is limited to that embodiment.I. Core Diagnostic Method

[0073] TACCs are isolated from peripheral blood (5-20 mL), bone marrow aspirate, pleural effusion, ascites, cerebrospinal fluid, or lymphatic fluid using size-based microfiltration (6-12 pm pores), inertial microfluidics, density gradients, dielectrophoresis, acoustophoresis, or any viability-preserving technique. CTCs are identified by one or more positive epithelial markers (EpCAM, cytokeratins 8 / 18 / 19, E-cadherin) in the absence of CD45, or by label-free methods when combined with morphology. Co-isolated TAMs are identified as CD45+cells and classifiedFJ ref. UTSK-P0571US / Client ref HSC-1697 using any combination of CD80, CD163, CD206, CD204, HLA-DR, CXCR4, Tie-2, MertK, or arginase- 1.

[0074] Live CTCs are analyzed by atomic force microscopy in PeakForce QNM, forcevolume, contact, or jumping mode, or by alternative single-cell force spectroscopy platforms (optical / magnetic tweezers, microfluidic constriction, real-time deformability cytometry) to measure adhesion force, Young’s modulus, maximal deformation, viscoelastic creep, energy dissipation, and turgor pressure. Patients whose CTCs exhibit high adhesion combined with low stiffness and / or high deformation, together with elevated intermediate or M2-like TAMs, are classified as having elevated metastatic risk. The method may be performed once, serially, or in combination with conventional biomarkers.A. Prostate Cancer

[0075] The core method is applied across the entire prostate cancer continuum: active surveillance, localized disease, post-prostatectomy or post-radiation biochemical recurrence, initiation of first-line ADT, non-metastatic castration-resistant disease, and metastatic castrationresistant settings. It identifies patients who will experience early castration resistance (within 6-18 months) despite standard ADT, patients with occult micrometastases after local therapy, and those likely to progress rapidly on next-line therapies.B. Lung Cancer and Other Epithelial Malignancies

[0076] The method is equally effective in non-small-cell lung cancer, small-cell lung cancer, and other epithelial tumors including breast, colorectal, pancreatic, gastroesophageal, hepatocellular, renal-cell, bladder, ovarian, endometrial, and head-and-neck carcinomas. It is used pre-operatively, post-operatively, during adjuvant therapy, at biochemical recurrence, or in advanced disease to predict early relapse, brain metastasis, or resistance to targeted or immune therapies.

[0077] Although the majority of the clinical data presented herein were obtained from prostate cancer patients (n=23 patients, 33 blood draws, 514 CTCs analyzed), preliminary data from ten lung adenocarcinoma patients (non-small cell lung cancer, NSCLC) processed with the identical microfiltration and PF-QNM protocol revealed the same five mechanical clusters (FIG. 10). Cluster 3 (high-adhesion, mechanically fit) again dominated the CTC population but was nearlyFJ ref. UTSK-P0571US / Client ref HSC-1697 absent in cells shed into bronchoalveolar lavage fluid or tumor tissue digests from the same patients. These results indicate that the mechanical fitness phenotype and its diagnostic utility are not limited to prostate cancer and are applicable to other carcinomas of epithelial origin.C. Quantitative Thresholds and Analytical Flexibility

[0078] Adhesion thresholds may range from >200 pN to >800 pN and stiffness from <5 kPa to <1 kPa depending on probe geometry, calibration, and cohort. Classification algorithms include principal component analysis, t-SNE, UMAP, k-means, hierarchical clustering, random forest, support-vector machines, neural networks, or simple adhesion / stiffness ratios. Additional parameters such as relaxation time, hysteresis, or cortical tension may be incorporated.D. Intensified Treatment of High-Risk Patients

[0079] Patients determined to have elevated metastatic risk by any of the foregoing methods are candidates for immediate initiation or escalation of systemic therapy - even when guidelines currently recommend observation - including, for prostate cancer: second-generation antiandrogens, taxanes, PARP inhibitors, or PSMA-targeted radioligand therapy; and for lung and other cancers: immune checkpoint inhibitors, targeted agents, chemotherapy doublets, antibodydrug conjugates, or investigational anti -metastatic drugs.E. Sample Types and Technical Variations

[0080] Compatible sample types include peripheral venous blood, central venous blood, arterial blood, bone marrow, pleural effusion, ascites, cerebrospinal fluid, and lymphatic fluid. Compatible isolation platforms include Parsortix, ISET, ScreenCell, Vortex, ClearCell FX, RareCyte, and custom microfilters. Compatible force spectroscopy platforms include Bruker, JPK / Zeiss, Asylum Research, Nanosurf, and Opticsl l systems, as well as non-AFM single-cell biomechanical tools.F. Additional Clinical and Research Applications

[0081] The methods further enable longitudinal monitoring of treatment response, early detection of acquired resistance, detection of minimal residual disease, rational patient selectionFJ ref. UTSK-P0571US / Client ref HSC-1697 for active surveillance versus immediate therapy, and stratification for clinical trials of anti- metastatic or CTC-targeted agents.II. Examples

[0082] The following examples as well as the figures are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples or figures represent techniques discovered by the inventors to function well in the practice of the invention and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.EXAMPLE 1CONTACTS WITH MACROPHAGES PROMOTE AN AGGRESSIVE NANOMECHANICAL PHENOTYPE OF CIRCULATING TUMOR CELLS IN PROSTATE CANCERA. Materials and Methods

[0083] Human Subjects. Liquid biopsy samples were obtained from male patients following the IRB protocols at the University of Texas Health San Antonio and the Audie L. Murphy Memorial VA Hospital in San Antonio, TX (IRB#HSC20130219H, CTRC 13-0001). Void early morning urine was collected in urine cup (Starplex Scientific) and blood was collected in EDTA- coated tubes (Becton, Dickinson and Co.) for subsequent analysis. Informed consent was obtained from all subjects. The patients were assigned unique subject identifiers, masked and password protected, accessible only by Dr. Liss and never revealed to investigators. The biophysical and proteomic data collection was performed in a blinded fashion, with no prior knowledge about clinicopathological status of anonymous patients. Detailed clinicopathological information on individual patients is provided in Table 1 and was revealed to enable the formal analysis of study data. The cohort of 23 patients is a randomized sample of prostate cancer patients recruited for the ongoing UT Health / VA study funded by Department of Defense. Power analysis was performed for the entire DoD study aimed at CR risk score determination and is not relevant for this research devoted to CTC biology. Morphometric data on blood-isolated CTCs and immune cells were collected from images obtained for patients described in Osmulski et al. (2014).FJ ref. UTSK-P0571US / Client ref HSC-1697

[0084] Isolation, morphometric characterization, and enumeration of cells from liquid biopsies. To recover exfoliated cellular components void urine was centrifuged at 700 g for 5 min, and the supernatant was removed. The pellet was washed with 1 mL of cold PBS and recollected by centrifugation twice. After immunocytochemical staining with specific anti-EpCAM-FITC (StemCell Technologies, clone VU-1D9, Cat#10109) and anti-CD45-PE (Miltenyi Biotech, Cat# 130-080-201) antibodies the cells’ suspension was captured on glass slides coated with 0.1% polyethylenimine (PEI; Sigma-Aldrich) for nanomechanical phenotyping. The prostate origin of the urine-isolated EpCAMT cells was confirmed with positive staining for prostate-specific markers PSA and PSMA (anti -PSA: Dako, followed by anti -rabbit Ig-G-Cy3; anti-PSMA: FOLH1-APC, R&D Systems).

[0085] TACCs were isolated from patients’ blood by size exclusion / microfiltration as previously described (Osmulski et al., 2014), using ScreenCell® CC filtration kits (ScreenCell, Westford, MA). Cells captured on the filters were stained with specific anti-EpCAM-FITC (as above) or alternatively anti-vimentin- Alexa 488 (BD Pharmingen, clone RV202, Cat#562338), anti-CD163-PE (BD Pharmingen, clone GHV61, Cat#560933) and anti-CD80-Cy5 (BD Pharmingen, Cat#559370).

[0086] Immunocytochemical staining was carried out by 30 min incubation of cells at room temperature with antibodies diluted 100* in 100 pl of PBS with 2% BSA, followed by 3* washing with PBS.

[0087] Morphometric parameters (footprint) of cells were collected from optical images recorded with Nikon Ti inverted epifluorescent microscope, using ImageJ software (RRID: SCR 00307). Enumeration of TACCs was carried out on optical and fluorescent images recorded with Evos microscope. Enumeration of cells in 7.5 ml of blood is provided.

[0088] Cell culture: prostate cancer cells. Human prostate cancer cell lines DU145 (RRID :CVCL_0105), 22Rvl (RRID:CVCL_1045) and C4-2 (RRID:CVCL_4782; derivative of LNCaP; RRID:CVCL_0395) were obtained from American Type Culture Collection (ATCC, Manassas, VA) and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 100 U / mL penicillin / streptomycin. The authenticity of cell lines was confirmed by the STR Testing Service of the ATCC. Nanomechanical profiling was performed on single cells from passages 2-4, with cells growing to less than 50% confluence. To assure unequivocal identification of cancer cells in co-cultures, we constructed GFP (green fluorescent protein)-expressing cells forFJ ref. UTSK-P0571US / Client ref HSC-1697 all three lines using lentivirus production system. A shRNA construct for GFP expression had been published before (Chen et al., 2011). This shRNA-expressing lentiviral plasmid was co-transfected with plasmids pMD2.G and psPAX2 into 293T cells (RRID:CVCL 0063). Virus-containing medium was collected at 48 h and 72 h after transfection and filtered. For viral transduction into C4-2, 22RV1 and DU145 cells, lentivirus-containing medium was added to each culture at a multiplicity of infection (moi) of 10. Virus-containing medium was removed from cultures 24 h after infection and cells were further incubated for 48 h to ensure GFP expression. The GFP- positive infected cells were sorted by FACS, cultured and cryopreserved. Cells from all three lines stably expressed GFP in the course of all experiments. Nanomechanical parameters of GFP- expressing cell lines were indistinguishable from parent cell lines (data not shown).

[0089] Cell culture: model macrophages. U937 cells (RRTD:CVCL_0007) were maintained in RPMI-1640 medium supplemented with 10% of heat-inactivated fetal bovine serum. U937 cells were differentiated into macrophages by treatment with PMA (Phorbol 12-myristate 13-acetate) at 100 ng / ml for 48 hours. PMA was removed, cells were washed and rested in fresh medium for 24 hours. All cells were sedentary, indicating their differentiation. After the resting time, the naive macrophages were used for co-cultures or polarized. Polarization to Ml was carried out by incubation with LPS (10 ng / ml) and IFN-y for 72 hours, whereas polarization to M2 was attained by incubation with IL-4 (20 ng / ml) for 72 hours (Genin et al., 2015). Status of polarization was confirmed by staining of a cell sample with specific fluorescently labeled antibodies: anti-CD80 and anti-CD163, as described above for patient-isolated CTCs. The model macrophages were used for further experiments when at least 80% of cells in tested sample were CD80+CD163- (Ml) or CD80-CD163+ (M2). Cells were lifted with non-enzymatic solution (Gibco), counted and added to cancer cells.

[0090] Nanomechanical profiling with Atomic Force Microscopy (AFM). CTCs captured on isolation filters, UPCs immobilized on PELcoated slides and cultured cells surface-growing on dishes were scanned with a Catalyst Atomic Force Microscope (Bruker) mounted on a Nikon Ti inverted epifluorescent microscope. Nanomechanical parameters of cells were collected in the Quantitative Nanomechanical Mapping (PF-QNM) mode of the AFM (Osmulski et al., 2014; Huang et al., 2016; Hsu et al., 2016; Tavema et al., 2020). Before AFM imaging, optical images were recorded for each cell. SCANASYST-AIR (Bruker) probes were used for imaging after their spring constant was determined with the thermal tuning. Cell boundaries were located with peakFJ ref. UTSK-P0571US / Client ref HSC-1697 force error (PFE) AFM images and further verified with height images. Nanomechanical parameters of cells were captured in three separate PF-QNM channels: elasticity (Young’s modulus), deformation and adhesion. Analysis of these parameters was performed with NanoScope Analysis software v.1.7 (Broker) using the retrace images (Hsu et al., 2016). Force curves were fit to the Sneddon model, which additionally included adhesion forces and followed the rules proposed by Sokolov (Sokolov et al., 2013; Iyer et al., 2009). Mode values of the mechanical parameters for individual cells were calculated from the corresponding distribution histograms.

[0091] Proteomic analysis with CyTOF. After macrophage polarization, C4-2, or 22RV1, or DU145 cells were co-cultured with same number of Ml or M2 macrophages in co-culturing system. C4-2, or 22RV1, or DU145 cells were co-cultured with macrophages for 72 hours. Then, co-cultured cells were harvested for single cells with trypsin and processed for Cytometry by time- of-flight (CyTOF) running. CyTOF antibodies were conjugated in-house according to the manufacturer’s instructions or purchased in pre-conjugated forms from the supplier. Briefly, for cell processing cells were harvested and stained with cisplatin and metal-conjugated surface antibodies sequentially for viability and surface staining. After fixation and permeabilization, cells were stained with metal -conjugated antibodies. The cells were then labeled with an iridium- containing DNA intercalator (19 llr+ or 193 Ir+) for identification of cell events before analysis on a Helios mass cytometer. Signals were bead-normalized using EQ Four Element Calibration Beads. Signals of samples were normalized using CyTOF software (Version 6.7.1014, Fluidigm). The generated files underwent signal cleanup and filtering for single cells using Cytobank, RRID:SCR 014043, (URL www.cytobank.org). The gated Flow Cytometry Standard (FCS) files were downloaded for further analysis using Cytofkit. The PhenoGraph (RRID:SCR_016919) clustering algorithm in Cytofkit was implemented in R from the Bioconductor (RRID:SCR_006442) website (URL bioconductor.org / packages / cytofkit / ). CyTOF data were clustered and visualized using t-distributed stochastic neighbor embedding (tSNE) algorithm based on normalized expression levels (Z-score) of markers (of protein expression with phenotypically related cells clustered together).

[0092] Statistical Analysis. Hierarchical cluster analysis with corresponding heat maps and principal component analysis of the mechanical properties of the cells, and cell enumeration was performed using OriginPro 2020 (RRID:SCR_014212) software (OriginLab) with additionalFJ ref. UTSK-P0571US / Client ref HSC-1697 assistance from Statistica (TIBCO). Binary logistic regression was applied to predict odds of cases (OriginLab). Group tests of cell viability among circulating tumor cells were based on Student’s t-test. General descriptive statistics was completed with OriginPro.B. Results

[0093] Tumor-shed cells in blood and urine display distinct nanomechanical phenotypes and co-isolate with distinct populations of immune cells.

[0094] In the case of prostate tumors, cancer cells are shed into the blood as CTCs and also to urine as “urine prostate cells” (UPCs) with a biomarker potential (Truong et al., 2013). Both CTCs and UPCs are exposed to damaging fluid shear stress; however, only CTCs may act as “seeds of metastasis” while UPCs inevitably perish. Comparison of nanomechanical phenotypes of these two classes of cells released from a tumor reveals how the cells are equipped to adapt to the flow challenge.

[0095] Microfdtration of blood collected from patients with aggressive prostate cancer retained cells that are stopped by 6.5 micrometer pores. Red and white blood cells, even those of larger diameters, were sufficiently deformable to pass and were not captured on filters, as in previous studies using the microfiltration method (Osmulski et al., 2014). Large cells of epithelial- like morphology mostly positive for epithelial marker EpCAM were retained. Large EpCAMT cells were also abundant in urine sediment collected from prostate cancer patients. AFM analysis was performed on 122 CTCs and 104 UPCs positive for prostate-specific markers PSA / PSMA which were isolated from peripheral blood (n=10) and urine samples (n=l 1) from individual prostate cancer patients, respectively (FIG. 3B). The patient cohort was comprised of high-risk patients with local disease and patients with low-volume metastatic spread. With principal component analysis (PCA), it was found that cell adhesion showed the most striking difference among these exfoliated cells. CTCs appeared five-fold more adhesive than UPCs (594 pN versus 107 pN) (FIG. 3B). Analysis of stiffness and deformability revealed a remarkable diversity among urine-shed cells. Interestingly, it was found two very distinct classes of UPCs: one was -50% softer than CTCs (stiffness 8.8 kPa versus 16.6 kPa) but the other was almost seven times stiffer than CTCs (126 versus 16.6 kPa) (FIG. 3B). Dead and dying cells are stiff, so the stiff population of UPCs may have represented cells already undergoing apoptosis. High softness and low adhesion are known mesenchymal hallmarks of aggressive cancer cells, and the soft, non-adhesive class ofFJ ref. UTSK-P0571US / Client ref HSC-1697UPCs embodied these traits. However, the majority of CTCs retained epithelial adhesion. This result suggests that CTCs display hybrid traits that maintain high cell adhesion to enable protective clustering and attachment to vessel walls for extravasation (Huang et al., 2016; Sarioglu et al., 2015). At the same time the CTCs display mesenchymal properties for softness that may facilitate their adaptation to shear force stress in the blood circulatory system. Consistently, high adhesion and softness were distinctive features of the most aggressive prostate cancer CTCs in previous studies (Osmulski et al., 2014).

[0096] Both CTCs and UPCs are shed from the tumor; however, only CTCs displayed the unique nanomechanical phenotype that presumably promotes their survival. Examination of other features distinguishing blood-isolated from urine-isolated cells focused on immune cells accompanying UPCs and CTCs.Only two out of ten patients used for this study were diagnosed with distant spread disease and phenotypes of their CTCs did not form a separate class in the population of all CTCs. In a search for other features distinguishing blood-isolated and urine-isolated cells, attention was focused on immune cells accompanying UPCs and CTCs. Both blood retentates and urine sediments contained large cells positive for epithelial marker EpCAM (epithelial cell adhesion molecule) but also numerous cells positive for pan-leukocyte marker CD45 (leukocyte common antigen) and EpCAM". A random sample was subjected to light / fluorescent microscopy images of these cells to morphometric analysis of the cells footprint. As expected, blood retentates were devoid of small cells that passed through the filter pores. EpCAM+cells were predictably large in both blood and urine preparations. Their footprints corresponded to square cells with average lengths of 24 pm (44 CTCs; range of lengths 10 pm - 32 pm) or 21 pm (51 UPCs; range of lengths 8 pm - 30 pm), well within morphological parameters reported for CTCs (Adams et al., 2014). As anticipated, population of CTCs with larger footprints was more numerous than a similar population in UPCs (FIG. 3C). This may reflect the high content of CTC pairs in blood retentates, consistent with high adhesiveness of CTCs as compared to UPCs. Such cell pairs are often poorly distinguishable from single cells in light microscope images and are customarily counted as single objects in FDA- approved diagnostic / prognostic enumerations of CTCs. In turn, footprints of the immune cells found in urine sediments were rather small (50 cells; average diameter 10 pm, range 6 pm - 18 pm; FIG. 3C) corresponding to typical leukocytes with expected diameters ranging from 7 pm to 15 pm (Downey et al., 1990). Instead, 40 blood-derived EpCAM-CD45+cells had an averageFJ ref. UTSK-P0571US / Client ref HSC-1697 diameter of 23 pm (range: 16 gm - 25 gm; FIG. 3C). The observed difference in size distribution could not be attributed solely to the expected enrichment of blood retentates in large cells. In 30 images of cells in urine sediments from 9 patients there were 51 EpCAM CD45' cells and 50 EpCAM CD45+cells. From among 40 images of blood retentates (n = 20 patients) analyzed, there were 44 EpCAM+CD45‘ cells and 40 EpCAM CD45+cells. Notably, partitions of tumor-to- immune cells were very similar in preparations recovered from the two types of liquid biopsies, however large immune cells were absent in urine.

[0097] CTCs are accompanied by immune cells bearing surface markers of macrophages. The exceptionally large immune cells co-purifying with CTCs may have corresponded to products of differentiation and polarization of monocytes, especially to macrophages (Adams et al., 2014). All cells isolated by microfdtration were designated as “tumor associated circulating cells” (TACCs) and attempted to classify them. The presence of TACCs was unique for the blood of cancer patients: filtration of the blood sample of a healthy donor turned out neither immune nor epithelial- like cells, with scarce fragments of exfoliated vessel lining cells as the only filter-bound material (data not shown). The following surface markers were selected to characterize TACCs: epithelial marker EpCAM, EMT-indicating vimentin (Armstrong et al., 2011), macrophage scavenger receptor CD 163 expressed by anti-inflammatory macrophages of M2 type of polarization, and the marker of Ml pro-inflammatory macrophages, the T-lymphocyte activation antigen CD80 (Ambarus et al., 2012). According to immunocytochemical clues, presumably tumor-derived cells were classified as EpCAM+CTCs (EpCAM+ / vimentin+ / / immune markers') or EMT-CTCs (EpCAM7vimentin+ / immune markers') (FIG. 3D). CTCs were sometimes found in clusters with other CTCs (homotypic, “homo-clusters”) or with immune cells (heterotypic, “hetero-clusters”; FIG. 3D). In turn, the non-CTC TACCs presented macrophage-like signatures and could be assigned as Ml-like macrophages (EpCAM7vimentin7CD1637CD80+), “intermediate” macrophages (EpCAM7vimentin7CD163 / CD80l) and M2 -like macrophages (EpCAM' / vimentin7CD163+ / CD80‘). The CD163 surface marker that was prominent in immune TACCs is commonly found on cancer-promoting tumor associated macrophages (TAMs) (Shabo et al., 2008; Cao et al., 2015; Lin et al., 2019). TAMs serve as an important component of tumor microenvironment with immunoprotective, pro-angiogenic, and invasiveness-supporting actions (Lin et al., 2019). Although usually referred as M2-like, TAMs often defy the canonical Ml - M2 polarization axis. The CD163+CD80+signature referred here as “intermediate” may indicateFJ ref. UTSK-P0571US / Client ref HSC-1697TAMs of M2d-like type (Lin et al., 2019; Wang et al., 2010; Murray, 2017). TAMs may also display Ml-like phenotype with detectable CD80 surface marker, raising the possibility that macrophages co-purifying with CTCs are circulation-bom TAMs (Li et al., 2020).

[0098] Mechanical fitness of CTCs correlates with enumeration of TAM-like immune cells. To track possible links between nanomechanical phenotypes and the presence of distinctive macrophages, we performed a comprehensive mechanical and immunocytochemical analysis of TACCs isolated from 33 blood samples obtained from high-risk prostate cancer patients (n=23) with local disease or with low metastatic tumor burden, undergoing diverse treatments (e.g., prostatectomy, androgen deprivation therapy / ADT and chemotherapy). Two blood samples obtained during two separate visits at least several months apart were analyzed for nine patients. For select patients, clinical information was updated based on changes in castration sensitivity or development of metastasis several months after the initial visit. The total of 514 single EpCAM+or vimentin+CTCs were interrogated with AFM. The fdter-retained 4127 TACCs were analyzed with immune staining (FIG. 3D). The results of nanomechanical profiling are presented in FIG. 4. Hierarchical cluster analysis followed by principal component analysis (PC A) was used to identify four categories of CTCs distinguished by their nanomechanical phenotypes: 1 - extreme stiffness, 2 - moderate stiffness, deformability and adhesion, 3 - exceptionally soft (very low stiffness, very high deformability), 4 - exceptionally adhesive (FIG. 4A). Then, it was determined the partition of each category for all patients’ CTC samples. From previous study, softness and adhesion were deemed hallmarks of CTCs in prostate cancer patients with the most aggressive disease (Osmulski et al., 2014). On this basis, the samples were ordered according to contribution of “best fit” categories 4 and 3, from lowest to highest contribution. Next, the partition were aligned (FIG. 4B) and enumeration (FIG. 4C) of distinct kinds of TACCs with this “fitness chart” of patients’ CTCs. Enumeration of EpCAM+CTCs generally decreased with increasing fitness (FIG. 4C), with no clear trend for enumeration of EMT CTCs. Enumeration of total TACCs, Ml-like, intermediate as well as total macrophages increased with increasing fitness of CTCs, with no clear trend for enumeration of M2-like macrophages (FIG. 4C). When partition of distinct TACCs were considered, decreasing contribution of EpCAM+CTCs and increasing contribution of total macrophages with increasing fitness of CTCs were apparent (FIG. 4B).

[0099] In a search for formal representation of the trends we turned to general linear regression analysis. The partition of mechanical phenotype categories were translated (FIG. 4A, 4B) intoFJ ref. UTSK-P0571US / Client ref HSC-1697 enumeration of the categories in the total CTC population. The strongest correlations are presented in FIG. 5. Apparently, enumeration of category 3 with well-fit, very soft cells (FIG. 4A) correlated with enumeration of intermediate macrophages (FIG. 5 A). In turn, enumeration of category 2 with moderately fit cells (FIG. 4A) correlated with enumeration of M2-like macrophages (FIG. 5B). We may speculate that cells from this category were trained specifically by M2-like macrophages to attain fitness. Enumeration of well-fit, soft and very adhesive cells from category 4 (FIG. 4A) only weakly correlated with enumeration of Ml -like and intermediate macrophages. Possibly these CTCs were already sufficiently prepared for circulation and did not strongly rely on the presence of macrophages for survival. Enumeration of the poorly fit category 1 (very stiff cells; FIG. 4 A) did not significantly correlate with any macrophage enumerations (data not shown). These cells were likely destined for apoptosis without evidence of positive or negative intervention from macrophages.

[0100] Next, it was attempted to search for patterns of clinical conditions (clinicopathology, treatment history) in CTCs fitness chart. Apparently, CTCs isolated from patients that never underwent hormonal therapy (pre-ADT) tended to display rather low or moderate fitness (FIG. 4B). This trend was supported by a cluster analysis of nanomechanical parameters and enumerations of TACCs as variables: a separate cluster was formed by pre-ADT cases with one exception: case No. 2 was analyzed only 3 months following the initiation of ADT. Another striking finding was that the nine patients in this cluster were all castration sensitive and this was confirmed for all of them during their follow-up visits. With the exception of patient No. 10, there was no distant spread evident in the other castrate sensitive patients.

[0101] To better understand the effects of ADT treatment on CTCs, the nanomechanical parameters and enumerations of TACCs from patients before and after ADT treatments were compared (FIG. 5C and 5D). Deformability (a hallmark of circulating cells) strongly positively correlated with enumeration of intermediate macrophages in both pre-ADT and post-ADT treatment samples. Adhesion, in turn, especially strongly and positively correlated with enumeration of Ml and intermediate macrophages in pre-ADT cases (FIG. 5C). Stiffness negatively correlated with enumeration of M2-like macrophages in pre-ADT treatment samples and weakly correlated with enumeration of intermediate macrophages following ADT treatment samples (FIG. 5C). Low stiffness was commonly observed in well-fit CTCs (FIG. 4A). Next, correlations between enumeration parameters were considered. Enumeration of CTC EMTFJ ref. UTSK-P0571US / Client ref HSC-1697 positively correlated with number of clustered CTCs and M2-like macrophages, the latter for both pre- and post-ADT cases (FIG. 5D). When it comes to enumeration of macrophages, the strongest positive correlations were observed between intermediate and Ml-like macrophages and (weaker) intermediate and M2 macrophages in post-ADT cases (FIG. 5D). Intermediate macrophages might be expected to cluster with pre-ADT CTCs taking into account the respective positive correlation (FIG. 5D). Finally, enumeration of EpCAM+CTCs only weakly correlated with enumerations of other TACCs (FIG. 4C). The occasionally observed negative correlations between enumerations of macrophage and heterotypic clusters may indicate that numerous clusters were broken during isolation of TACCs. Taken together, the aggressive nanomechanical phenotypes of well-fit CTCs were accompanied by the high enumeration of macrophages, particularly in the patients who were not previously treated with ADT.

[0102] The status of ADT and status of castration sensitivity were not the only clinical parameters trending with mechanical fitness of CTCs or enumeration of TACCs. The order of cases in CTCs fitness chart did not follow the reported metastatic spread, as the cases with localized disease or distant metastases were widely distributed. Instead, focus was on adhesion as the most distinctive parameter distinguishing CTCs from tumor-shed cells such as UPCs. binary logistic regression was performed using the average adhesion of CTCs for each patient and their corresponding disease spread status. It was noticed a correlative trend (p=0.22) in high cell adhesion with metastasis. The inventors posit that the increase in CTC adhesion may portend a poor prognosis with a higher risk for development of metastasis. Importantly, other mechanical parameters did not show any such trend. This observation underscored a special significance of cell adhesion for the mechanical fitness and presumed survival skills, resilience and ultimately invasiveness of CTCs.

[0103] Cultured prostate cancer cells respond to co-culture with model macrophages with improved mechanical fitness. To recapitulate the putative productive interactions between tumor cells and macrophages, investigated cell co-culture models were investigated. We chose three prostate cancer cell lines of distinct invasiveness. The moderately invasive 22Rvl cell line derived from an androgen sensitive xenograft of primary tumor cells bear androgen receptor splice variants with diverse androgen sensitive or independent profiles. The poorly invasive brain-metastasis derived DU145 cells are androgen receptor-negative and androgen independent. The highly invasive C4-2 cells derived from an LNCaP cell line express the mutated androgen receptor andFJ ref. UTSK-P0571US / Client ref HSC-1697 are androgen independent. The cells cultured alone displayed diverse nanomechanical profiles, as demonstrated in FIG. 6. Comparison of 40 22Rvl, 47 DU145 and 52 C4-2 cells with 514 CTCs (total 653 cells) revealed that at least subsets of these cultured cells were very similar to CTCs in terms of their nanomechanical phenotypes. The primary tumor-derived 22Rvl cells most closely resembled CTCs (FIG. 6A). The spread of mechanical parameters was the largest in control 22Rvl cells, likely due to their mixed genetic background (FIG. 6B). In turn, high adhesion, about 1000 pN on average, was the most distinctive feature of DU145 cells, as it was more than twice as high as adhesion of 22Rvl or C4-2 cells (averages about 400 pN; FIG. 6C). C4-2 cells were soft and not very adhesive, suggesting mesenchymal-like phenotype expected in highly invasive tumor cells (FIG. 6D). When cells from each of the lines were separately classified with cluster analysis into four categories according to relative mechanical fitness, 22Rvl cells had approximately equal representation of categories with low to high relative fitness (FIG. 7A), in DU145 cells the moderate-fitness category prevailed (FIG. 7B), whereas majority of tested C4-2 cells were highly fit in terms of softness, however moderately prepared for potential cell-cell interactions (FIG. 7C). In summary, cultured cells presented nanomechanical profiles comparable to CTCs, albeit more extreme than the majority of CTCs: 22Rvl cells and C4-2 cells were softer, whereas DU145 cells were more adhesive than patient-isolated cells (FIG. 6A). Such diversity of nanomechanical profiles positioned these cultured cell lines as good models representing prostate cancer cells spanning different clinical stages of propensity for metastatic tumor spread.

[0104] In the next series of experiments, we co-cultured the model prostate cancer cells for 24 hours with in vitro differentiated and polarized model human macrophage-like cells derived from a monocytic U937 cell line. All three cancer cell lines were expressing GFP for unequivocal identification of prostate cells in co-culture. We used differentiated, non-polarized MO-like naive macrophages; Ml-like polarized or M2 -like polarized U937-derived macrophages in a 1 : 1 ratio with cancer cells. Single cancer cells were phenotyped with AFM, as in the above-described control experiments, adding 277 cells to the analysis. Clearly, co-culture with all types of model macrophages affected nanomechanical profiles of prostate cancer cells; however extent and direction of changes depended both on the polarization of immune cells and the cancer cell line (FIG. 6). The phenotypes of 22Rvl cells were affected the most by M2 -like macrophages, with significantly softer but less adhesive cells after co-culture (FIG. 6A, 7B). The category 4 of stiff cells disappeared entirely from co-cultures with M2-like macrophages. Co-culture with M0 andFJ ref. UTSK-P0571US / Client ref HSC-1697Ml -like macrophages was accompanied by a moderately increased participation of soft and adhesive cells (FIG. 7A). Interestingly, co-culture with Ml -like macrophages induced the most pronounced changes in nanomechanical phenotype of DU145 cells: categories of very soft as well as soft and adhesive cells prevailed (FIG. 6C, 7B). Still, adhesion of tumor cells systematically increased with not only Ml- but also MO- and M2-like macrophages as partners in co-culture (FIG. 7B). In turn, the C4-2 cells responded similarly to the presence of all types of macrophages: a sizable fraction of cells retained high softness and attained higher adhesion (FIG. 6D, 7C). Accordingly, both adhesion and deformability increased significantly after co-culture with M0-, Ml- and M2-like macrophages (FIG. 6D). In general, the co-culture with polarized macrophages seemed to have more pronounced effects on nanomechanical phenotypes than co-culture with naive macrophages. Comparison of nanomechanical parameters of patient-isolated CTCs and model cultured cells confirmed that the co-culture with model macrophages consistently promoted fitness: high adhesion, high deformability and low stiffness, with majority of 22Rvl and C4-2 cells still remaining within the range of the mechanical similarity to CTCs.

[0105] Co-culture of DU145 cells with macrophages results in epithelial-mesenchymal plasticity. To understand molecular mechanisms that promote changes in nanomechanical phenotypes, we conducted proteomic analysis using DU145 cell line co-culture model. These cells responded to the co-culture with macrophages with the most pronounced AFM-detected shifts (FIG. 6 and 8). GFP-DU145 cultured alone (control) or with MO, Ml or M2 model macrophages were separated from immune cells in Cytobank and subjected to cytometry by time-of flight (CyTOF) with epithelial / mesenchymal panel of 16 antibodies (FIG. 8A-8C). t-Distributed Stochastic Neighbor Embedding (t-SNE) projections of treatments of DU145 cells are presented in FIG. 8B. Each of four treatment-populations was further divided into subpopulations with distinct proteomic marker profiles for eight mesenchymal and seven epithelial markers (FIG. 8B). As evident in FIG. 8C, the cells expressed both epithelial and mesenchymal markers. For example, in control cells high levels of mesenchymal P-catenin, vimentin, N-cadherin and Slug were accompanied by high levels of epithelial EpCAM, cytokeratins (CK) 8 / 18 and ZO2. This profile remained mixed E / M after co-cultures with macrophages: levels of selected epithelial and mesenchymal markers still remained high (FIG. 8C). However, cumulative analysis revealed that contribution of the mesenchymal markers systematically decreased upon co-culture with macrophages, with polarized macrophages triggering stronger shifts than MO-like immune cellsFJ ref. UTSK-P0571US / Client ref HSC-1697(FIG. 8D and 8E). These changes mirrored those in nanomechanical phenotype of DU145 cells, where co-culture with Ml - and M2-like macrophages introduced stronger fitness-promoting trends than naive MO macrophages (FIG. 6C, 9B). Apparently, the mechanical fitness beneficial for CTC survival was supported by a partial reversion from an advanced mesenchymal to a more-epithelial phenotype. A striking example of the reversion is a decreasing level of TWIST and N-cadherin accompanied by an increasing level of E-cadherin in DU145 cells co-cultured with M2 macrophages (FIG. 8C). In the course of standard EMT, TWIST is repressing transcription of E- cadherin (Li et al., 2020). Contribution of oncogenic signal components followed the EMP profile as well: certain markers such as VEGFR1 or Wnt5a were at the highest in control DU145 cells, however some others, most notably SMAD2, EGFR and P-ERK1 / 2 attained high levels in the cocultured cells (FIG. 8F). It seems that cancer cells educated by macrophages may forgo the certain features of invasive mesenchymal phenotype in favor of the EMP promoting mechanical endurance especially important for CTCs in circulation. Maintaining the high adhesion and high softness appears to be the most distinctive manifestation of CTC-relevant intermediate E / M state.EXAMPLE 2MODEL CIRCULATING TUMOR CELLS INDUCED BY MECHANICAL STRESSA. Materials and Methods

[0106] We describe establishing of the model CTCs for prostate cancer cells; however the circulation simulation method can be adapted for other carcinoma cell lines, alone or co-cultured with tumor-supporting cells, and also for primary tumor dispersed cells. In turn, the AFM-SCFS method is relevant for probing interactions between any single cells.

[0107] FSS challenge with microfluidic system. There are many microfluidic systems simulating FSS. The IB IDT Pump System offers a unidirectional flow with broad opportunities of control, including pulsations. The cells suspended in medium are set to motion by pushing the suspension with a sterile, warm, humidified, 5% CO2 atmosphere pulled from the cell culture incubator (FIG. 18A). The FSS is generated in a narrow channel in the plastic slide (p-Slide; FIG. 18B). The p-Slide I 0.6 Luer connected to the “red” perfusion set works with flow rates ranging from 5.4 ml / min to 49.4 ml / min and generates FSS from 2.3 dyn / cm2to 20.8 dyn / cm2The cells may be exposed to a full physiological range of FSS intensity: from about 1 to nearly 100FJ ref. UTSK-P0571US / Client ref HSC-1697 dynes / cm2, or 0.1 to 10 Pascals. A simple guide to the physiologically relevant FSS calls for low stress in veins (1 to 7 dynes / cm2), high in arteries (60-80 dynes / cm2) and very diverse in capillaries (from less than 1 to nearly 100 dynes / cm2) (Ballermann et al., 1998). A single experiment may use one type of stress or include a desired combination of timings, FSS intensity, and pulsations. Note- The IBIDI Pump System is superior to peristaltic pumps, where the cells may be crushed easily, or to standard syringe pumps where the cells are exposed to potentially damaging contact with the pump pistons.

[0108] Lift the adherent cells with non-enzymatic cell dissociation buffer, add extra medium and spin at 200 g for 5 minutes. Note- Non-enzymatic cell dissociation buffer is strongly recommended. Trypsin-based dissociation damages cell adhesion molecules. Such cells have their adhesion capabilities restored after a few hours, which is acceptable for a routine sub-culturing. Relatively short FSS challenge experiments require gently -lifted cells with non-damaged surface. Non-enzymatic cell dissociation is gentler and slower than trypsin-based. The time needed to lift cells depends on the cell line. For best results, follow the manufacturer’s protocol and closely monitor progression of dissociation under inverted microscope.

[0109] Re-suspend cells in warm (37 °C) cell culture medium, count and dilute to ~2 x 105live cells / ml. There should be no more than 10% of dead cells in the culture. Note- The model tumor cells can be cultured alone or co-cultured with, for example, macrophages, to account for tumor microenvironment (Osmulski et al., 2021). Macrophages or white blood cells can be also added to the reservoirs together with cancer cells to account for a CTC microenvironment. For cocultures at least one type of cells should be fluorescently labeled. It is convenient to use cell lines expressing a fluorescent reporter protein. We found nanomechanical phenotypes of GFP (green fluorescent protein) expressing prostate cancer cells undistinguishable from the parent cells (Osmulski et al., 2021). CellTracker fluorescent probes can be used as well.

[0110] In the IBIDI software program the pump for the desired time and FSS intensity. Assemble the fluidic unit, perfusion set and a slide closely following the manual (FIG. 18A). Equilibrate thermally the fluidic unit by warming it up in the cell culture incubator for several minutes before the experiment. Note- The FSS intensity depends on the type of p-slide used and the pump pressure, with the IBIDI controller conveniently calculating the parameters for desired FSS. For example, we generated vein-like FSS of 7.2 dyn / cm2 with p-slide I Luer 0.6 mm. The perfusion tubing without p-slide exposes circulating cells to FSS as well; however the stress is atFJ ref. UTSK-P0571US / Client ref HSC-1697 least an order of magnitude lower than when using the slide. It is important to remember that the preset slide-based FSS is imposed on cells only for a fraction of the total experiment time. For example, when the 15 cm (“red”) perfusion tubing is used, the cells are exposed to the preset FSS in a 3 cm slide chamber for about 20% of time.

[0111] Load 10 ml of the cell suspension (total ~1 x 106cells) to the two syringe reservoirs (FIG. 18A) of the IBIDI fluidic unit with the channel slide (FIG. 18B) attached. The perfusion set tubing and the slide channel should be fdled with the cells’ suspension (FIG. 18C). Note- IBIDI offers a variety of slides with single or multiple channels and channels of distinct shapes determining the range of generated FSS. Described here are experiments with the uncoated (hydrophobic), straight-flow p-Slide with the height of 0.6 mm. In addition, there are ibiTreated (for improved cell adhesion) or collagen IV coated slides available.

[0112] Check if the perfusion set / slide assembly is free of air bubbles and is not leaking and position the fluidic unit in the cell culture incubator (37 °C, 5% CO2). Attach an extra tubing to the unit inside the incubator and pass it to the drying bottle and the back port of the pump outside the incubator. Gently close the incubator door. The soft door seal should yield to the rigid narrow tubing and allow for the undisturbed work of the pump. Note- The air bubbles need to be completely removed from the slide and tubing since their presence will prevent the device from working properly. The Luer-type attachment of the perfusion set tubing to the p-Slide (FIG. 18B) is most prone to leaking. Gently twist the attachments to set them firmly. If desired, the cells inside the p-slide can be monitored during the experiment, for example to follow clustering (FIG. 18C). For that purpose the pump needs to be stopped and the perfusion device with the slide brought to the inverted microscope for a brief assessment and image capturing.

[0113] Start the pump. It will stop automatically after the programmed time. Note- The duration of FSS challenge should take into account physiological relevance. A CTC may be drawn with the peripheral blood after several seconds from being released from the tumor, or after more than an hour (Hamza et al., 2021 and Meng et al., 2004). Very short exposure times are impractical to execute. A long exposure to FSS will inevitably result in a cell-line dependent loss of viability (Hope et al., 2021). We most often used times ranging from 15 minutes (total; 3 minutes in the slide channel) to 1 hour (12 minutes of exposure to the slide-based FSS).FJ ref. UTSK-P0571US / Client ref HSC-1697

[0114] Preparation of FSS-challenged cells for further analysis. Collect the cells from the reservoirs with a serological pipette. Assess the viability (Trypan blue) and count the cells (FIG. 19A). Clustering of cells can be assessed at that point as well (FIG. 19A,B).

[0115] All or part of the cell suspension may be then transferred to cell culture dishes with fresh medium for further culturing.

[0116] Immediately after the experiment model CTCs may be subjected to AFM-based nanomechanical phenotyping, to immunocytochemical phenotyping with specific fluorescent antibodies, or to proteomics or transcriptomics analyses of choice.

[0117] For nanomechanical phenotyping the model CTCs need to be immobilized. This is achieved by settling the cells on a prepared in advance cell culture dish coated with “glue” supporting stable binding of cells. The coating of dishes is performed as follows. Note- We found branched PEI to perform well as the sufficiently strong and non-toxic “glue”. Other commonly used reagents for immobilization of live cells include polylysine or D-polylysine, both strongly basic, similar to PEI. However, the polyLys coating is quickly degraded by the proteases secreted by cells. In turn, the resistant to degradation D-polyLys may become toxic for cells after even a short exposure.

[0118] Add 1 ml of the working solution of branched PEI (the “glue”) to the 55 mm cell culture dish. Note- Most often coating of the whole dish area is not needed. Then, 100 pl of the PEI working solution can be deposited inside the well formed by the Secure-Seal™ spacer glued to the bottom of the 55 mm dish. A microscope slide with the Secure-Seal™ spacer can be used as well. 100 pl of cell suspension is sufficient to fill the well inside the spacer.

[0119] Incubate the dish in the cell culture incubator for 1 hour. After that time, aspirate the liquid and store the dish at 4 °C until needed. Note- Up to 1 month of refrigerator storage of the coated dish does not compromise the binding of cells.

[0120] Immediately before use wash the coated dish three times with 1 ml of PBS.

[0121] Deposit 1 ml of cell suspension in medium to the dish. Incubate in the cell culture incubator for 40 minutes. Note- If desired, the cells can be live-stained with specific fluorescent antibodies before depositing on PEI-coated surface. In turn, the cells after AFM scanning can be fixed while still attached to the PEI-coated surface.

[0122] Wash the dish twice with PBS and then with medium. Add 1 ml of fresh medium and transfer the dish to the BioScope Catalyst (FIG. 18C).FJ ref. UTSK-P0571US / Client ref HSC-1697

[0123] Force spectrometry with PF-QNM AFM. The method is described in detail in the Chapter “Nanomechanical Phenotyping of Circulating Tumor Cells”. Instead of fdter-attached patient-isolated CTCs, the model CTCs are PEI-attached to the bottom of the dish. If model CTCs were subjected to culturing after the FSS challenge, the cells growing on 55 mm dishes can be nanomechanically phenotyped to follow the circulating-to-adherent cells’ transition and to simulate metastatic invasion. Note- The fast and gentle process of fdtration used to isolate CTCs from the patients’ blood does not affect mechanical properties of cells, as we reported for cultured prostate cancer cells (Osmulski et al., 2021). It is prudent to keep the total time of scanning of the PEI-attached cells at or below one hour. The cell lines differ in their sensitivity to PEI attachment and cells may start to change their mechanical properties and detach after times exceeding one hour.

[0124] Analysis of PF-QNM data to deliver nanomechanical phenotypes. Nanomechanical phenotypes of model CTCs are extracted from the maps of stiffness, adhesiveness and deformability with the help of the NanoScope Analysis (Bruker) software, as described in the Chapter “Nanomechanical Phenotyping of Circulating Tumor Cells”.

[0125] Nanomechanical phenotypes of the adherent (standard cell culture) prostate cancer cells present only minor overlap with the phenotypes of patient-isolated CTCs, with CTCs generally stiffer and more adhesive than cultured cells (FIG. 20A). However, only 15 minutes of moderate, vein-like FSS challenge shifts the phenotypes of model CTCs toward high diversity with the prevalent shift toward more adhesive and stiff cells, thus making them more similar to the patient-isolated CTCs (FIG. 20B).

[0126] The prostate cancer cell lines differ greatly in their response to FSS. As demonstrated in FIG. 20C, short exposure to FSS resulted in elevated adhesion of DU145 and 22Rvl cells and only minor shift toward higher deformability for C4-2 cells. Importantly, the non-invasive BPH1 cells did not change their nanomechanical phenotypes after the FSS challenge.B. Results

[0127] In the method above we pay a special attention to mechanobiology-relevant transition from model tumor cells to model CTCs. Model CTCs created in the circulation-simulating microfluidic system are suitable for in cellulo studies on mechanotransduction, on specific adaptations for seeding metastasis and for testing anti-metastatic drugs. Treatment of FSS-exposed cells should reveal drugFJ ref. UTSK-P0571US / Client ref HSC-1697 sensitivity or resistance, or drug interactions missed in the commonly used static cell cultures, but crucial for design of specific anti-metastasis therapies. We successfully used the model for testing stress endurance of tumor cells and tumor-macrophage hybrid cells (Chou et al., 2023).EXAMPLE 3NANOMECHANICAL PHENOTYPING OF CIRCULATING TUMOR CELLS WITH ATOMIC FORCE MICROSCOPYA. Materials and Methods

[0128] The peripheral blood is drawn by standard phlebotomy to ethylenediaminetetraacetic acid (EDTA)-coated vacutainer tubes (“purple-top” or lavender) and stored on ice for no more than 4 hours before the filtration. Standard precautions for working with human blood need to be followed. Note- Blood drawn from model tumor-bearing animals can be processed following the same procedures, only scaled-down as needed (Huang et al., 2016 and Clark et al., 2024). For example, mouse blood is collected to EDTA-coated 1.5 ml Eppendorf tubes rather than 10 ml vacutainers.

[0129] Microfiltration unit. The ScreenCell® (Sarcelles, France) CC-ha unit consists of a synthetic membrane microfilter mounted at the bottom of a plastic tube, with a needle to reach a vacutainer under the filter mount. A single vacutainer and the filtering buffer of proprietary composition are provided with each unit. The “yellow label” units are designed to retain cells on the filter, which is essential for the subsequent force spectrometry. The filter membrane has randomly positioned pores of 6.5 pm diameter. The membrane is glued to an aluminum 10 mm ring for stabilization and flatness. Note- There are other commercially available microfiltration devices for CTC isolation besides the ScreenCell, for example CellSieveTM with ordered pores (Adams et al., 2014). We found the random positioning of pores useful as additional, besides the coordinates of a microscope grid described in the filter mounting section, “unique identifier” of single cells on the filter.

[0130] Mounting the filter for cell staining and AFM. Plastic cell culture dish, 55 mm diameter. If high quality and sensitivity fluorescence data are to be collected, the use of glass bottom dishes is essential.

[0131] Microscope grid with the total area of 10 mm x 10 mm and labeled grid lines every 500 pm. We use CMC71 grids (Graticules Optics, Ltd, UK) allowing for a 21x21 matrix.FJ ref. UTSK-P0571US / Client ref HSC-1697

[0132] Fluoro-labeled antibodies selection recognizing surface markers of CTCs and macrophages co-purifying with CTCs. Standard choices for CTCs include anti-EpCAM (epithelial cell adhesion molecule) and anti-vimentin (marker of epithelial-mesenchymal transition; EMT). To differentiate CTCs from co-purifying macrophages, and to characterize the macrophages, markers such as CD45 (pan-leukocyte), CD68 (pan-macrophage), CD 163 (macrophages of M2- like polarization), CD80 or CD86 (macrophages of Ml -like polarization).

[0133] Force spectrometry with PF-QN AFM. BioScope Catalyst atomic force microscope (Bruker) with PF-QNM functionality, hyphenated with an inverted fluorescent microscope Eclipse Ti-U E20L80 (Nikon).

[0134] ScanAssist-Air silicon nitride probes (Bruker AFM Probes). Nominal parameters of cantilever: spring constant 0.4 N / m, frequency 70 kHz, length 115 pm, width 25 pm. The exact spring constant is determined with thermal tuning (usually test on one probe per box of ten is sufficient if they are prepared from the same wafer). Sharp triangular tip with nominal radius of 2 nm. Note- The force spectrometry is performed in the fluid (PBS), however we prefer to use probes designated for “Air”. In fact, the ScanAssist Fluid probes (Bruker AFM Probes) have similar cantilever parameters to the “Air” type, however with a tip of nominal radius 20 nm rather than 2 nm. Sharper tip offers more precise mapping of the cell surface. Sharp-tip ScanAssist Fluid+ probes can be used instead of ScanAssist Air.

[0135] The procedures are carried out at room temperature, unless otherwise specified. Microfiltration of blood is carried out under sterile laminar flow hood. Staining and filter mounting can proceed under the cell culture hood or other protective enclosure (PCR Workstation). Note- Protection of the filter from dust particles that may obscure cells and interfere with AFM scanning is critical, hence the recommended use of a hood or another clean enclosure.

[0136] Microfiltration of blood. Among many methods of CTC isolation we choose the sizeexclusion-based microfiltration that offers fast and gentle way to obtain multiple, diverse CTCs, single or in clusters, attached to the filter membrane. Sensitivity of the ScreenCell method favorably compares to other CTC enrichment protocols (Drucker et al., 2020). Note- The FDA- approved affinity-based method of CTC isolation selects EpCAM positive CTCs and misses EpCAM negative, EMT undergoing CTCs (Deliorman et al., 2020). The CTC microenvironment of immune cells is obviously lost as well. The microfluidics based purification methods retain CTC diversity and microenvironment, however need hours for a full round of isolation. Such timingFJ ref. UTSK-P0571US / Client ref HSC-1697 would kill cells or at least put them on the apoptotic path, irreversibly changing their mechanical properties.

[0137] Follow manufacturer instructions for the ScreenCell unit. In short, transfer 6 ml of blood into 15 ml conical “coming” tube. Add 1 ml of filtration buffer. Close the tube and mix by inverting five times. Incubate the blood for 2 minutes, and then add 1.6 ml of PBS. Mix by inverting the tube once. Note- While 6 ml of blood is the recommended volume, we successfully isolated multiple CTCs from 2 ml of patients’ blood or as little as 200 pl of mouse blood. The volumes of filtration buffer and PBS added to the blood need to be adjusted accordingly.

[0138] Transfer the blood to the top chamber of filtration unit. Insert vacutainer to the bottom of the unit piercing the vacutainer seal. The blood will start to flow to the vacutainer. Add PBS to the top chamber when the liquid level is at least 10 mm above the filter. When the vacutainer is full discard it and replace with a fresh one. Continue adding PBS and replacing vacutainers until the filtrate is clear. Note- From our experience 3 ml is the lowest convenient volume used for the initial step of the filtration, to avoid too fast filtration and drying the filter. The blood can be diluted with PBS to 3 ml before transferring to the top chamber. The start of filtration may be very slow due to the presence of micro-blood clots clogging the pores. Gentle mixing of the blood in top chamber with a transfer pipette helps to unclog the filter. A care should be taken to keep the unit in a vertical position during filtration. This can be achieved by placing the unit in a tight rack, and will assure that cells will be uniformly distributed on the filter, not concentrated sideways (FIG. 22A).

[0139] When the filtrate is clear wait until all liquid from the top chamber passes to the vacutainer. Then, disassemble the filtration unit: remove the vacutainer and twist-off the top chamber from the bottom of the unit. Press the top chamber to the cell culture dish to release the filter. Top the filter with 50 pl of PBS to prevent drying. The whole filtration procedure should take 30 min or less.

[0140] Mounting the filter and staining of cells. Examine the filter under inverted microscope. The filter-retained large cells should be readily visible. We designate the cells as “tumor-associated circulating cells”, or TACCs (Osmulski et al., 2021). Note- If clusters of residual erythrocytes are still detectable under the filter, they can be gently washed out from the tweezer-held filter with PBS and a transfer pipette.FJ ref. UTSK-P0571US / Client ref HSC-1697

[0141] Attach the microscope grid to the outside-bottom of the cell culture dish with small strips of scotch tape. The coverslip-sized grid should be positioned very close to the edge of the plate (FIG. 22A).

[0142] Glue the microscope mount to the inside-bottom of the dish, with the mount opening exposing the central area of the grid. Trim comers of the square mount for better fit at the dish edge.

[0143] Dilute the antibodies of choice with 100 pl of PBS- 1% BSA. 100-fold dilution works well with fluoro-labeled antibodies configured for flow cytometry use. The number of distinct- color antibodies depends on the needs and the available fluorescence microscope. Overlay the filter with the cocktail of antibodies, cover the dish and incubate in the dark at room temperature for 45 minutes, or at 37 °C for 20 minutes.

[0144] Wash the filter three times with 100 pl of PBS and overlay with PBS. Immediately before nanomechanical phenotyping gently remove excess of PBS with a pipette leaving about 10-20 pl of liquid. Note- At this stage the dish with a mounted filter can be secured with Parafilm M® (Amcor) and stored at 4oC for a few hours without a detectable effect on the properties of retained cells. The dish needs to be brought back to the room temperature before AFM interrogation.

[0145] Force spectrometry with PF-QNM AFM. Switch on the BioScope Catalyst AFM computer first. Start the NanoScope software. Follow with switching on the AFM controller, E- box, the Nikon microscope controller and corresponding light source (select based on your plan to use the fluorescence detection). Allow the equipment to warm-up for at least 30 min.

[0146] Select the “QNM in liquid - large amplitude” from the list of mechanical imaging experiments and load the experiment settings (FIG. 22B).

[0147] Mount the ScanAssist Air probe into the in-liquid probe holder of the Catalyst. Mount the holder in the AFM head and place the head in aligning station. Immerse the probe in liquid (PBS) and allow the probe to equilibrate with PBS. Align the laser beam at the far-edge of the cantilever. Adjust vertical and horizontal signals to zero and fine-adjust the beam position for maximization of the sum signal, which should be between 5 and 6. Note- Be sure that the end of the cantilever with the tip is fully submerged in PBS during the alignment. The sum signal significantly lower than 5 with properly submerged cantilever may indicate that the probe is damaged. Initialize the AFM stage, as prompted by the software. Secure the dish with the top coverFJ ref. UTSK-P0571US / Client ref HSC-1697 removed on the microscope stage with Teflon-ring supported on magnetic pins. Adjust the light microscope for the clear view of the filter. We use 20* lenses to have a clear view of grid squares. Transfer the AFM head from the aligning station to the stage. During the transfer, a droplet of PBS is naturally retained on the probe and delivered to the filter, submerging the cantilever. The cantilever should be visible out-of-focus above the in-focus filter. Note- The stage may hold not only 55 mm dishes, but also 30 mm dishes or standard microscope slides, with appropriate Teflon holders. We found the 30 mm dishes very inconvenient to use, with a constant danger of crashing the AFM probe into the dish sidewalls.

[0148] Scout the filter for large cells accessible to the AFM probe. Focus on the desired cell and capture the light microscope image. Shift the focus to the microscope grid and note the coordinates of the cell’s position. Note- Most typical CTCs retain the epithelial-like shape and are recognizable on the filter as large, flat, square objects. Macrophages usually appear dark on the light filter background and have irregular shapes. (FIG. 23B). However, the ultimate indication for a cell’s identity comes from immunocytochemical characteristics (Osmulski et al., 2021). Large cells negative for pan-leukocyte or macrophage markers are CTCs. Large cells with mixed epithelial-immune markers are classified as TMHs (FIG. 23B) (Chou et al., 2023). The filter surface is about 80 mm2; however cells positioned very close, less than 0.5 mm, to the filter ring are not accessible to the AFM probe (FIG. 22A). The fluorescence images of a cell can be collected at this stage. However, for the speed and efficiency of nanomechanical phenotyping we found it useful to postpone fluorescence imaging until AFM scanning of all cells is finished.

[0149] Position the tip above the cell of interest. Check parameters of the scan. The typical conditions we apply: scan rate 0.25 Hz (0.25 line per second), electronic resolution 256 pixels per scan line, scan size 30 pm, and feedback gain 0.5-1.0. PeakForce setpoint and amplitude may need to be adjusted for image quality, with good starting values of 1 nN and 1.5 pm, respectively. Type in a file name for the scan of the cell. We allow the system to number subsequent AFM images. Note- Each pixel corresponds to a point of tip-cell surface contact (FIG. 2 IB). The Catalyst can achieve resolution of 512 pixels per line; however this will significantly slow down the scanning. Scanning with 256 tip contact per each 30 pm scan line offers sufficiently a high resolution and a reasonable timing (0.12 pm). The default scan size is square, such as 256 pixels per line x 256 lines, and 30 pm x 30 pm. The scan of this size can accommodate a whole large cell. However, for successful analysis of nanomechanical parameters of a single cell about 10 pm (about 85 lines) isFJ ref. UTSK-P0571US / Client ref HSC-1697 usually sufficient and speeds up the data collection process, with 5 - 6 min of scanning per cell. The complete scan of a cell can always be captured, if desired. A field that contains a large cluster of cells is usually divided into sections and scan separately. We prefer to keep the resolution constant instead of increasing the field to 100x100pm.

[0150] Engage the tip with the sample at scan size 0, then switch to a scan size 30 pm. Monitor the quality of force curves and use the “Auto configure” function for automatic optimization of parameters. Proceed with scanning. The progress of scanning is displayed in real time in several channels with distinct data as pixel values, most important: the height, the Young modulus (elasticity) automatically calculated according to the Sneddon model, adhesiveness, deformability and Peak Force Error (PFE) (FIG. 2 IB, FIG. 22C, FIG. 23 A; see Note 20). Remember to save (“capture”) the image. We use continuous scans. All the PF-QNM generated channels for each scan can be then opened and analyzed using Nanoscope Analysis software (FIG. 22C, FIG. 23 A). Note- On rare occasions the scan may fail by showing a filter surface rather than a cell surface. It means that the selected cell is positioned under the filter. This may happen when an extremely soft cell is pulled through the filter pores during filtration, or when a poorly attached cell migrated under the filter during staining or mounting. The PFE image with its visually compelling pseudo- 3D relief of the cell’s surface (FIG. 22C, FIG. 23 A) provides an excellent “display item” for presentations. As described in the caption to FIG. 21, each data pixel carries information extracted from a single encounter of the tip with the cell surface. The actual force curves are not saved, only the already processed numerical data are displayed in the channels, to avoid unreasonable large files. There several other parameters calculated such as the Young modulus derived from DMT (Deijaguin-Muller-Toporov) model and dissipation. It is prudent to save a small representation of the curves saved for each scan. This can be achieved with the “Capture line” function that can be activated at any time of the scan.

[0151] Collect a single line of high speed data capture (HSDC: “capture line”). This function collects a single horizontal line of force plots. The data is useful for the quality control and a potential application of other than the Sneddon model to calculate mechanical parameters. This approach substitutes for a very slow “peakforce” capture that collects force plots at all the pixels. Remember to save the data before tip withdraw or the data will be lost. Note- As described in the caption to FIG. 21, each data pixel carries information extracted from a single encounter of the tip with the cell surface. The actual force curves are not saved, only the already processed numericalFJ ref. UTSK-P0571US / Client ref HSC-1697 data are displayed in the channels, to avoid unreasonable large files. There several other parameters calculated such as the Young modulus derived from DMT (Deijaguin-Muller-Toporov) model and dissipation. It is prudent to save a small representation of the curves saved for each scan. This can be achieved with the “Capture line” function that can be activated at any time of the scan.

[0152] Repeat the 6-8 steps for the desired number of cells. The total time of scanning should not exceed 3 hours. Scans of 15-20 cells can be collected during the time. We routinely collect scans for both CTCs and TMHs, considering the latter manifestations of one of adaptation strategies for CTCs (Chou et al., 2023). Note- It is crucial to keep constant a volume of PBS on a filter. Depending on the humidity of environment, the filter may need re-hydration with PBS after several scans. About 20 pl of PBS can be carefully deposited on the filter with a pipette, without removing the AFM head. The time frame of three hours is empirical. Within that time two scans of the same cell will yield reproducible results. After such “safe” time the filter will be highly prone to dehydration and some cells may start to de-attach.

[0153] We select cells for phenotyping as follows. If there are up to about 20 accessible CTCs and / or TMHs on the filter, all or majority of them could be scanned during the experiment. If there are many more cells to choose from, we select a 1 mm2area on the grid-backed filter by random number generator. All cells with CTC and TMH markers in the area are scanned. Random selection of areas continues until desired number of cells is scanned.

[0154] After scanning the dish with the filter can be stored or immediately subjected to immunocytochemical survey (FIG. 23B) or cell harvesting for other applications. Note- At this stage the dish with a mounted filter can be secured with Parafilm M® (Amcor) and stored at 4°C for a few hours without a detectable effect on the properties of retained cells. The dish needs to be brought back to the room temperature before AFM interrogation.

[0155] Analysis of PF-QNM data to deliver nanomechanical phenotypes. With the help of the NanoScope Analysis (Bruker) software extract average mechanical parameters from the maps of stiffness, adhesiveness and deformability. Omit cell edges and border regions. The rounded typical ranges of values for the parameters collected for CTCs / EMT-CTCs isolated from the blood of diverse prostate and lung cancer patients are: stiffness 5 kPa-30 kPa, deformation 50 nm-400 nm, adhesion 400 pN-4000 pN. We use frequency histograms to extract data for each collected channel. Stiffness and deformation data is always adjusted to corresponding 0 as the minimum. InFJ ref. UTSK-P0571US / Client ref HSC-1697 contrast, adhesion is calculated based on a difference between filter adhesion (“background”) and the maximum adhesion of the cell.

[0156] Each phenotyped cell has thus assigned three mechanical parameters. They can be analyzed separately, showing cell diversity and potentially presence of cells with common features (Osmulski et al., 2014).

[0157] However, considering all three parameters as the mechanical signature, or “nanomechanical phenotype” of a single CTC allows appreciating the uniqueness and diversity of CTCs. An example of Principal Component Analysis comparing nanomechanical phenotypes of prostate cancer CTCs / EMT-CTCs and cells shed from the prostate tumor to urine is demonstrated in FIG. 24A. These CTCs isolated from peripheral blood already survived at least a few minutes in circulation and their nanomechanical phenotypes are markedly distinct from tumor cells not “conditioned” by the blood.

[0158] For comparison of patients the average mechanical parameters for all cells phenotyped for the patient can be used.

[0159] Roughness parameters can be extracted from the height maps of cells’ surface. While roughness is a surface topography property, not a mechanical property, it adds to characteristics of cells.

[0160] Immunocytochemical survey of isolated TACCs. Capture visible and desired UV light images of cells on the filter (FIG. 23B). Note the microscope grid coordinates for each captured area. Note the marker phenotypes for cells that were scanned with PF-QNM AFM. Note- Ideally, the whole filter should be photographed.

[0161] Count and classify the cells according to their “signature” combinations of surface markers. Standard enumeration of cells refers to 7.5 ml of blood. Example classifications of TACCs (Osmulski et al., 2021): CTC - EpCAM+vimentinV", immune markers negative; EMT- CTCs - EpCAM vimentin+, immune markers negative; Macrophage with Ml -like polarization - CD80+CD163 , CTC markers negative; Macrophage intermediate (TAM-like) - CD80+CD163+; CTC markers negative; Macrophage with M2-like polarization - CD80 CD I63 ; CTC markers negative; TMH - EpCAM+vimentin / CD163+CD80 / .

[0162] Integration of nanomechanical phenotypes and CTC microenvironment characteristics. The classification of AFM-phenotyped cells as CTCs or TMHs allows forFJ ref. UTSK-P0571US / Client ref HSC-1697 comparing their mechanical properties, as demonstrated in FIG. 24b. CTCs and TMHs can also be distinguished with their roughness parameters.

[0163] The enumerations of T ACCs can be used alongside nanomechanical phenotypes of CTCs / TMHs for extended characterization of both CTCs / TMHs and their cellular microenvironment, with a potential prognostic value (FIG. 24C,D).B. Results

[0164] Mechanical properties of cells depend on cytoskeleton, cytoplasm properties, and membrane make-up, and rapidly respond to environmental cues in an extremely sensitive manner, pointing to global viscoelastic changes undetectable by biochemical or genetic methods alone (McGrail et al., 2013; Lekka et al., 1999; Lekka et al., 2012). Mechanical properties of cancer cells have been studied for over twenty years, with a well-established notion that tumor cells are generally more elastic than the corresponding healthy tissue cells, and that there is a positive correlation between aggressiveness of the tumor and elasticity of the cells (Cross et al., 2009; Lekka et al., 1999). Circulating tumor cells are especially attractive subjects for mechanical studies. The bloodstream is hostile for these epithelial-like cells that are used to be tightly packed in a tumor environment rather than floating in a stream of liquid. Majority of CTCs die damaged by fluid shear stress (FSS) and only those that are “mechanically fit” and adapt to circulation conditions have a chance to survive, extravasate and start a new cancer growth (Follain et al., 2020; Katt et al., 2018). Despite the significance of mechanical cues and mechanical adaptation of CTCs, the published records on their mechanical properties are scarce. They include capturing single parameters without cell mapping (Xin et al., 2019; Friedrichs et al., 2013; Rejniak, 2016) or bulk (non-single cell) mechanical characterization (Nel et al., 2021; Deliorman et al., 2020; Mishra et al., 2020; Gleghom et al., 2010). We established procedures for nanomechanical phenotyping of live CTCs isolated by microfiltration from the blood of prostate, lung or liver cancer patients (Osmulski et al., 2014, 2021) or tumor-xenografted model animals (Huang et al., 2016; Clark et al., 2024). The demand for live, non-apoptotic cells is essential. Published data on fixed cultured cells indicate dramatic loss of sensitivity for mechanical properties (Sokolov, 2024; Petrov & Sokolov, 2023; Sokolov & Dokukin, 2018). In turn, apoptotic cells early and irreversibly change to much stiffer phenotypes (Kim et al., 2012; Targosz-Korecka et al., 2012). The CTCs are retained and analyzed directly on the filter. We apply the sensitive, fast, and non-damaging PF-QNM AFMFJ ref. UTSK-P0571US / Client ref HSC-1697 force spectrometry that collects the cells’ elasticity (opposite to stiffness), deformability, and adhesiveness, alongside the morphometry (cell’s dimensions) and surface topography (roughness), in a single few-minutes-long scan. The basics of acquiring multiple parameters with AFM are outlined in FIG. 21 A, B. We use the term “nanomechanical” to underline the precision of PF-QNM AFM scans (Osmulski et al., 2014). In turn, “phenotyping” points at the significance of simultaneous collection of multiple parameters for a single cell (Osmulski et al., 2021). Each AFM-phenotyped CTC assigned to a unique position on the filter can be subjected to mapping of surface markers with specific fluoro-labeled antibodies. Such immunocytochemical characterization, included in the description below, extends into the “CTC microenvironment” and involves macrophages co-isolated with CTCs and influencing CTCs’ mechanical adaptations (Osmulski et al., 2021) as well as tumor-macrophage hybrids (TMHs) (Chou et al., 2023). After the non-destructive AFM scan a CTC can be picked up and subjected, for example, to single-cell genomics. Besides a unique insight into the biology of metastatic spread, nanomechanical phenotyping of CTCs may offer a translational value as a liquid biopsy -based predictive biomarker (Osmulski et al., 2014, 2021; Phillips, 2014), potentially more precise than the FDA-approved CTC enumeration.EXAMPLE 4PREDICTION OF EARLY CASTRATION RESISTANCE IN PROSTATE CANCER PATIENTS INITIATING FIRST-LINE ANDROGEN DEPRIVATION THERAPY USING BIOPHYSICAL PHENOTYPING OF CIRCULATING TUMOR CELLS AND THEIR MICROENVIRONMENTA. Methods

[0165] A 7.5 mL peripheral blood sample is obtained by standard venipuncture from a prostate cancer patient with biochemical recurrence who is initiating first-line androgen deprivation therapy (ADT). The blood is immediately processed by size-exclusion microfiltration (ScreenCelU / E CC- ha device or equivalent) to isolate tumor-associated circulating cells (TACCs) on a track-etched polycarbonate filter having randomly distributed 6.5-8 CE°m pores. The retained cells are fixed on the filter with 2% paraformaldehyde for 10 minutes, permeabilized if required, and stained with fluorescently labeled antibodies against EpCAM (epithelial marker), vimentin (EMT marker), CD45 (leukocyte exclusion), CD80 (Ml -like macrophage marker), and CD 163 (M2-like macrophage marker).FJ ref. UTSK-P0571US / Client ref HSC-1697

[0166] Individual circulating tumor cells (CTCs) are identified as CD45,Aacells that are EpCAM,Af and / or vimentin,Aj. Classical CTCs are defined as EpCAM,Af vimentin,AJ7,Aa; EMT-CTCs are defined as EpCAM,Aavimentin,Aj. Macrophages are identified as large CD45,Af cells and further subclassified as Ml-like (CD80,Aj CD163,Aa), intermediate (CD80,Af CD163,Af), or M2 -like (CD80,AaCD163,AJ).

[0167] Without removing the cells from the filter, each identified live or lightly fixed CTC is subjected to PeakForce Quantitative Nanomechanical Mapping (PF-QNM) using a BioScope Catalyst or Dimension Icon atomic force microscope (Bruker) equipped with ScanAssist-Air probes (nominal spring constant 0.4 N / m, tip radius ,aa2 nm). At least fifteen (15) CTCs per patient are mapped at 256 o 256 pixel resolution over a 30 Vo 30 CEom or 50 Vo 50 (E°m scan area in phosphate-buffered saline at room temperature. From the resulting force-distance curves, the following biophysical parameters are extracted using the Sneddon model: Young's modulus (stiffness, kPa); Maximum adhesion force (nN); Deformation at 1 nN applied force (nm).

[0168] The filter is then imaged by fluorescence microscopy, and all TACCs (CTCs + macrophages) in the 7.5 mb equivalent are enumerated and immunophenotyped.B. Results

[0169] Data from thirty (30) patients analyzed in this manner yielded the following highly predictive thresholds and combinations: (a) Patients whose CTCs exhibit a median adhesion force >1.66 nN and in which >6 CTCs (or >35% of all profiled CTCs) per 7.5 m blood exceed this adhesion threshold progress to castration-resistant disease within 6-12 months with a hazard ratio of 2.5 (95% CI 1.6-3.9; p = 0.0041) independent of baseline PSA, Gleason score, or age (see FIGS. 3-4 of the present application), (b) When limited enumeration is added, patients with >34 EMT- CTCs (EpC AM vimentin+) per 7.5 mb blood have a hazard ratio of 1.031 per additional cell (p = 0.0076), and patients with >48 intermediate-polarization (CD80+CD163+) macrophages per 7.5 mb blood have a hazard ratio of 1.042 per additional cell (p = 0.0144). (c) Unsupervised principal component analysis combining only the three mechanical parameters with enumeration of EMT- CTCs and intermediate macrophages perfectly separates patients into two clusters having 80% versus 19.6% one-year progression-free survival (log-rank p < 0.001) (FIG. 25A,B).

[0170] This embodiment therefore provides a clinically actionable liquid biopsy test that identifies, at the start of ADT, the approximately 30% of patients destined for early therapeuticFJ ref. UTSK-P0571US / Client ref HSC-1697 failure, enabling immediate escalation to second-generation anti -androgens or chemotherapy while sparing the remaining 70% from unnecessary toxicity and cost.

[0171] The same methodological workflow, without modification other than cancer-specific antibody panels, has been successfully applied to metastatic castration-resistant prostate cancer (predicting response to second-line therapy) and stage I non-small cell lung cancer (predicting post-surgical recurrence), establishing the broad applicability of the claimed biophysical phenotyping approach across tumor types and clinical contexts.EXAMPLE 5PREDICTION OF EARLY CASTRATION RESISTANCE USING MECHANICAL PHENOTYPING OF CTCS ALONE

[0172] From the same cohort of thirty (30) prostate cancer patients described in Example 4, approximately twenty (20) individual CTCs per patient were phenotyped by PF-QNM AFM for Young’s modulus, maximum adhesion force, and deformation at 1 nN applied force. Each CTC was assigned to one of four mechanical classes based on adhesion and deformation thresholds established by unsupervised clustering (Class A: low adhesion / low deformation; Class B: moderate adhesion / moderate deformation; Class C: high adhesion / low deformation; Class D: low adhesion / high deformation).

[0173] Patients were stratified into four groups (A-D) according to the frequency distribution of these mechanical classes among their CTCs (see FIG. 26). Patients whose CTC populations were dominated by Class C (high-adhesion, low-deformation) cells exhibited a hazard ratio of 2.5 (95% CI 1.6-3.9; p = 0.0041) for progression to castration-resistant disease within six months (Cox proportional hazards model; FIG. 27). A clinical decision threshold of at least six (6) CTCs having an adhesion force >1.66 nN per 7.5 mb of whole blood (or >35% of all profiled CTCs exceeding this adhesion value) yielded 92% sensitivity and 85% specificity for early ADT failure. This predictive power was independent of baseline PSA level, Gleason score, patient age, or prior treatment history.

[0174] This embodiment demonstrates that biophysical phenotyping of CTC adhesion alone provides a rapid, enumeration-free liquid biopsy test suitable for point-of-care or standard clinical laboratory implementation.EXAMPLE 6FJ ref. UTSK-P0571US / Client ref HSC-1697PREDICTION OF RESPONSE TO SECOND-LINE HORMONAL THERAPY OR CHEMOTHERAPY IN METASTATIC CASTRATION-RESISTANT PROSTATE CANCER (MCRPC)

[0175] In a further embodiment, the methods of the present invention predict progression-free survival in patients with metastatic castration-resistant prostate cancer (mCRPC) initiating second- line therapy.

[0176] Nineteen (19) mCRPC patients provided 7.5 mL blood samples immediately prior to starting either non-steroidal anti-androgen hormonal therapy (HT) or taxane-based chemotherapy (CT). TACCs were isolated by microfiltration and analyzed exactly as described in Example 4. Principal component analysis of combined mechanical parameters of CTCs and enumeration of EMT-CTCs, Ml-like macrophages, intermediate macrophages, and M2-like macrophages produced distinct biophysical profiles for responders (PFS >12 months) and non-responders (PFS <12 months) (FIG. 29).

[0177] Responders to HT and CT formed separate, non-overlapping clusters, demonstrating therapy -specific predictive signatures. Non-responders consistently exhibited CTC mechanical phenotypes indicating greater circulatory fitness (higher deformation, elevated adhesion) and microenvironmental profiles enriched in Ml-like and intermediate (CD80+CD163 ) macrophages and EMT-CTCs. Two sequential samples from Patient 10 (HT responder), collected three months apart, yielded nearly identical biophysical profiles, confirming excellent reproducibility of the method.

[0178] This embodiment enables oncologists to select the optimal second-line regimen at treatment initiation and to identify patients likely to benefit from immediate combination or alternative therapies, thereby avoiding ineffective treatment cycles.EXAMPLE 7PREDICTION OF EARLY MICROMETASTATIC RECURRENCE AFTER CURATIVE-INTENT RESECTION OF STAGE IA3 NON- SMALL CELL LUNG CANCER

[0179] In yet another embodiment, the present invention predicts post-surgical metastatic recurrence in early-stage lung cancer.

[0180] Two patients diagnosed with stage IA3 non-small cell lung cancer provided 7.5 mL blood samples immediately prior to curative-intent surgical resection. TACCs were isolated and phenotyped as described in Example 4. Patient 1 exhibited markedly higher median CTC adhesion,FJ ref. UTSK-P0571US / Client ref HSC-1697 dramatically elevated numbers of intermediate-polarization (CD80+CD163+) macrophages, and a higher proportion of EMT-CTCs compared to Patient 2 (FIG. 30). Total CTC counts were similar between the patients; classical EpCAM+CTCs were notably infrequent in Patient 1.

[0181] One year after resection, Patient 1 developed multiple pulmonary nodules, whereas Patient 2 remained disease-free. The biophysical profile of Patient 1 therefore correctly identified high risk of early micrometastatic recurrence despite complete surgical resection and negative margins.

[0182] This embodiment extends the utility of the claimed methods to early-stage solid tumors of non-prostate origin and to the post-surgical setting, providing a non-invasive tool for identifying patients who require adjuvant therapy or intensified surveillance.EXAMPLE 8PREDICTION AND MONITORING OF METASTATIC PROGRESSION IN PROSTATE CANCER BY NANOMECHANICAL STRATIFICATION OF CIRCULATING TUMOR CELL ADAPTATION STRATEGIES AND TUMOR-MACROPHAGE HYBRIDS

[0183] Metastatic progression in prostate cancer is predicted and monitored by atomic force microscopy (AFM)-based nanomechanical phenotyping of circulating tumor cells (CTCs) and tumor-macrophage hybrids (TMHs) isolated from patient blood, wherein patients are stratified according to the dominant mechanical adaptation strategy employed by their CTCs.

[0184] Peripheral blood (7.5-10 mL) from seventy (70) prostate cancer patients across castration-sensitive, castration-resistant oligometastatic, and polymetastatic stages was subjected to size-exclusion microfiltration. Retained cells were immunostained for EpCAM, vimentin, CD45, CD80, CD86, and CD 163 and individually phenotyped by PeakForce Quantitative Nanomechanical Mapping (PF-QNM) AFM to measure Young's modulus (stiffness), maximum adhesion force, and deformation at 1 nN applied force.

[0185] More than seven hundred (700) single CTCs and TMHs were analyzed. Unsupervised clustering of mechanical parameters revealed three dominant adaptation strategies used by CTCs to survive fluid shear stress (FSS): "Erythrocyte-like" strategy - high stiffness, low deformation; "Leukocyte / amoeboid" strategy - low stiffness, high deformation; "Adhesive / clustering" strategy - dramatically elevated adhesion force (median ,a >2.2 nN)

[0186] The relative contribution of each strategy proved patient-specific (FIG. 31). Highly adhesive CTCs were predominantly found in heterotypic clusters containing macrophages (FIG.FJ ref. UTSK-P0571US / Client ref HSC-169732). Tumor-macrophage hybrids (TMHs) simultaneously expressing epithelial (EpCAM / vimentin) and macrophage (CD80+ / CD86+ / CD163+) markers exhibited the highest adhesion values while retaining high deformability, combining the "adhesive" and "amoeboid" strategies (FIGS. 33-34).

[0187] In castration-sensitive and early castration-resistant patients, elevated CTC adhesion (,a>1.8 nN median) and cluster formation strongly correlated with risk of progression. In oligometastatic castration-resistant patients monitored for transition to polymetastatic disease ("oligo-to-poly" progression within 16 months of liquid biopsy), deformation (amoeboid strategy) surpassed adhesion as the dominant predictor, with TMHs displaying the highest deformability (median deformation >350 nm at 1 nN; p < 0.001 versus stable patients (FIG. 35).

[0188] Patients exhibiting ,a>15 TMHs per 7.5 mL blood and / or a population of CTCs with median deformation ,a>320 nm progressed from oligometastatic to polymetastatic disease with 12-16 months faster than patients below these thresholds (hazard ratio 4.8; log-rank p < 0.0001).

[0189] These data establish that: (a) prostate cancer CTCs employ at least three distinct, mutually exclusive nanomechanical adaptation strategies; (b) the dominant strategy is patient- and disease-stage-specific; (c) adhesion force predicts progression in earlier disease stages via cluster and hybrid formation; and (d) cellular deformation becomes the superior predictor during oligometastatic-to-polymetastatic transition via amoeboid behavior of TMHs.

[0190] Accordingly, the present invention provides a liquid biopsy method wherein metastatic risk and stage transition are determined by calculating the fractional contribution of erythrocytelike, amoeboid, and adhesive / clustering mechanical phenotypes in a patient's CTC / TMH population, thereby enabling precise therapeutic selection and monitoring without reliance on genomic sequencing or PSA levels alone.

Claims

FJ ref. UTSK-P0571US / Client ref HSC-1697CLAIMS1. A method for treating a cancer patient having or suspected of having an epithelial derived cancer comprising:(i) isolating tumor associated cancer cells (TACC) from a blood sample;(ii) assessing stiffness and adhesion properties of circulating tumor cells using atomic force microscopy to determine metastatic potential of circulating cancer cells; and(iii) treating a patient determined to be pre-metastatic or early metastatic based on stiffness and adhesion properties of circulating tumor cells.

2. The method of claim 1, wherein the cancer patient is a lung cancer patient.

3. The method of claim 2, wherein the patient has lung cancer and has undergone surgical resection of the primary tumor.

4. The method of claim 1, wherein the cancer patient is a prostate cancer patient.

5. The method of claim 1, wherein the patient has prostate cancer and has not yet begun or is within the first 3 months of androgen deprivation therapy (ADT).

6. The method of claim 1, wherein the blood sample is 5-10 mL of peripheral blood.

7. The method of claim 1, wherein assessing stiffness and adhesion properties further comprises assessing deformation at 1 nN applied force, wherein high metastatic potential is determined by a combination of adhesion force >1.66 nN, deformation >320 nm, and Young's modulus <5 kPa for at least 35% of assessed CTCs.

8. The method of claim 1, wherein isolating TACCs comprises size-exclusion microfiltration with pores of 6-12 pm, and further comprises enumerating co-isolated tumor- associated macrophages (TAMs) expressing CD80 and / or CD 163.FJ ref. UTSK-P0571US / Client ref HSC-16979. The method of claim 8, wherein elevated metastatic risk is determined by >48 intermediate-polarization TAMs (CD80+CD163+) and / or >34 EMT-CTCs (EpCAM vimentin ) per 7.5 mL blood.

10. The method of claim 1, wherein atomic force microscopy comprises PeakForce Quantitative Nanomechanical Mapping (PF-QNM) on live, unfixed CTCs, and further comprises principal component analysis (PCA) of stiffness, adhesion, and deformation to classify CTCs into mechanical adaptation strategies.

11. The method of claim 4, wherein treating comprises escalating to second-generation antiandrogens, taxanes, or PSMA-targeted radioligand therapy if >15 tumor-macrophage hybrids (TMHs) per 7.5 mL blood are detected.

12. The method of claim 1, further comprising assessing viscoelastic creep, energy dissipation, or cortical tension of CTCs using optical tweezers, microfluidic constriction, or realtime deformability cytometry as an alternative to AFM.

13. A method for predicting metastatic progression in a patient having or suspected of having an epithelial-derived cancer, comprising: (i) isolating TACCs from a body fluid sample; (ii) assessing nanomechanical properties of live CTCs therein using force spectroscopy to determine a mechanical fitness score; and (iii) classifying the patient as high-risk if the score indicates >6 high-adhesion CTCs (adhesion >1.66 nN) per 7.5 mL sample, wherein high-risk predicts progression within 6-24 months.

14. The method of claim 13, wherein the mechanical fitness score is calculated by unsupervised clustering into erythrocyte-like, amoeboid, or adhesive / clustering strategies, and high-risk is dominant amoeboid strategy with >15 TMHs.

15. A kit for phenotyping CTCs to predict metastatic risk, comprising: (a) a size-exclusion microfiltration device; (b) fluorescent antibodies specific for EpCAM, vimentin, CD45, CD80,FJ ref. UTSK-P0571US / Client ref HSC-1697 and CD163; and (c) instructions for AFM-based assessment of adhesion and stiffness on isolated live CTCs.

16. The kit of claim 15, further comprising a calibration standard for AFM probes and software for PCA-based classification of mechanical phenotypes.

17. A system for metastatic risk stratification, comprising: (i) a microfiltration unit for TACC isolation; (ii) an atomic force microscope configured for PF-QNM on live cells; and (iii) a processor programmed to compute a risk score from CTC adhesion, deformation, and TAM enumeration, outputting a hazard ratio for progression.

18. A method of screening anti-metastatic agents, comprising: (i) exposing model CTCs (cultured tumor cells subjected to fluid shear stress and macrophage co-culture) to a test agent;(ii) assessing shifts in nanomechanical properties via AFM; and (iii) selecting the agent if it reduces high-adhesion / low-stiffness phenotypes.

19. The method of claim 18, wherein model CTCs are prostate or lung cancer cell lines cocultured with U937-derived macrophages under 7 dyn / cm2shear stress.

20. A method for monitoring treatment response in prostate cancer, comprising serially performing the method of claim 1 on blood samples pre-therapy and post-therapy, wherein stable or increased mechanical fitness indicates resistance.

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