Single-cell profiling of phagocytosis using microscopy

Abstract

The invention relates to methods of evaluating phagocytosis at a single-cell level by placing one or more phagocytes and one or more particles on a surface and visualizing the phagocytosis of the particles by the phagocytes. The invention also relates to systems for evaluating phagocytosis of a particle by a phagocyte at a single-cell level.

Description

PCT Application Attorney Docket No. AF23853.P209WOUH ID No. 2024-089TITLESINGLE-CELL PROFILING OF PHAGOCYTOSIS USING MICROSCOPYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims priority from, and incorporates by reference the entire disclosure of, U.S. Provisional Patent Application 63 / 722,535, filed on November 19, 2024.BACKGROUND

[0002] A need exists for more effective methods and systems for evaluating phagocytosis. Numerous embodiments of the present disclosure aim to address the aforementioned need.SUMMARY OF THE INVENTION

[0003] In some embodiments, the present disclosure pertains to methods of evaluating phagocytosis at a single-cell level by placing one or more phagocytes and one or more particles on a surface and visualizing the phagocytosis of the particles by the phagocytes. In some embodiments, the methods of the present disclosure also include steps of characterizing one or more molecular profiles of the phagocytes and integrating the visualized phagocytosis and the molecular profiles of the phagocytes. In some embodiments, the methods of the present disclosure also include a step of implementing a treatment decision based on the evaluation.

[0004] Additional embodiments of the present disclosure pertain to systems for evaluating phagocytosis of a particle by a phagocyte at a single-cell level. In some embodiments, such systems include a non-transitory processor operable for evaluating visualized phagocytosis of a particle by a phagocyte. In some embodiments, the processor may include programming instructions for integrating the visualized phagocytosis and molecular profiles of phagocytes. In some embodiments, the processor may also include programming instructions for recommending a treatment decision based on the evaluation.

[0005] In some embodiments, the systems of the present disclosure may also include a surface operable for receiving one or more phagocytes and one or more particles. In some embodiments, the systems of the present disclosure also include a sensor operable for visualizing the phagocytosis of the particles by the phagocytes.DESCRIPTION OF THE DRAWINGSPCT Application Attorney Docket No. AF23853.P209WOUH ID No. 2024-089

[0006] FIG. 1A illustrates a method of evaluating phagocytosis at a single-cell level.

[0007] FIG. IB illustrates a system for evaluating phagocytosis at a single-cell level.

[0008] FIG. 2 provides micrographs showing THP-1 cell (unstained) opsonizing beads manufactured with DragonGreen dye. coated with CD 19 spike protein and pHrodo dye (Red). The beads were incubated with antibody against the spike protein before a TIMING assay.

[0009] FIG. 3 provides micrographs showing antibody mediated phagocytosis of B16 mouse melanoma cells recombinantly expressing HER2 and (unstained) cells by THP-1 derived macrophages (Red). B16 cells were incubated with Trastuzumab antibody.DETAILED DESCRIPTION

[0010] It is to be understood that both the foregoing general description and the following detailed description are illustrative and explanatory, and are not restrictive of the subject matter, as claimed. In this application, the use of the singular includes the plural, the word “a” or “an” means “at least one”, and the use of “or” means “and / or”, unless specifically stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements or components comprising one unit and elements or components that include more than one unit unless specifically stated otherwise.

[0011] The section headings used herein are for organizational purposes and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated herein by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials defines a term in a manner that contradicts the definition of that term in this application, this application controls.

[0012] Phagocytosis is a key process in the innate immune response. When phagocytes arrive at the site of an infection or a tumor, they ingest the foreign pathogen or apoptotic cell. Once consumed, the phagocytes can then activate the lymphocytes by presenting antigens bound by major histocompatibility complexes (MHC).

[0013] Phagocytes can also prevent the spread of cancer cells by destroying them. However, many tumors develop strategies to evade the immune mechanism.PCT Application Attorney Docket No. AF23853.P209WOUH ID No. 2024-089

[0014] As an emerging therapeutic method, boosting phagocytosis is being used to eliminate cancer cells. However, previous methods and technologies aimed at evaluating and monitoring phagocytosis are mostly based on cytometry-based methods, such as flow cytometry assays. While cytometry-based assays are very effective at providing snapshots of cellular phenotypes, they are not well suited for studying continuous dynamic cellular behaviors or time-lapse microscopy of a limited number of manually sampled “representative” cell pairs (e.g., 10-100). As such, prior methods of evaluating phagocytosis are inherently biased and cannot quantify the behaviors of statistically under-represented cells.

[0015] Accordingly, a need exists for more effective methods and systems for evaluating phagocytosis. Numerous embodiments of the present disclosure aim to address the aforementioned need.

[0016] In some embodiments, the present disclosure pertains to methods of evaluating phagocytosis at a single-cell level. In some embodiments illustrated in FIG. 1A, the methods of the present disclosure include: placing one or more phagocytes and one or more particles on a surface (step 10); and visualizing the phagocytosis of the particles by the phagocytes (step 12). In some embodiments, the methods of the present disclosure also include steps of characterizing one or more molecular profiles of the phagocytes (step 14); and integrating the visualized phagocytosis and the molecular profiles of the phagocytes (step 16). In some embodiments, the methods of the present disclosure also include a step of implementing a treatment decision based on the evaluation (step 18).

[0017] Additional embodiments of the present disclosure pertain to systems for evaluating phagocytosis of a particle by a phagocyte at a single-cell level. With reference to system 20 in FIG. IB for illustrative purposes, system 20 includes a non-transitory processor 26 operable for evaluating visualized phagocytosis of a particle by a phagocyte. In some embodiments, processor 26 may include programming instructions for integrating the visualized phagocytosis and molecular profiles of phagocytes. In some embodiments, processor 26 may also include programming instructions for recommending a treatment decision based on the evaluation.

[0018] In some embodiments, system 20 may also include a surface 22 operable for receiving one or more phagocytes 23 and one or more particles 24. In some embodiments, system 20 also includes a sensor 25 operable for visualizing the phagocytosis of the particles 24 by the phagocytes 23. In some embodiments, sensor 25 may be on surface 22.PCT Application Attorney Docket No. AF23853.P209WOUH ID No. 2024-089

[0019] In some embodiments, processor 26 may also include a receiver 27 operable for receiving phagocytosis-related images, such as from surface 22. In some embodiments, receiver 27 is a camera. In some embodiments, system 20 may also include a display 28 operable for displaying the visualized phagocytosis.

[0020] As set forth in more detail herein, the methods and systems of the present disclosure can have numerous embodiments and applications.

[0021] Phagocytes

[0022] The methods and systems of the present disclosure may be utilized to evaluate the phagocytosis of various phagocytes. For instance, in some embodiments, the phagocytes include, without limitation, plant cells, fungi cells, bacterial cells, prokaryotic cells, eukaryotic cells, unicellular cells, multi-cellular cells, immune cells, tumor cells, or combinations thereof. In some embodiments, the phagocytes include, without limitation, T cells, B cells, monocytes, macrophages, neutrophils, dendritic cells, natural killer cells, fibroblasts, stromal cells, stem cells, progenitor cells, tumor cells, tumor stem cells, tumor infiltrating lymphocytes, macrophages, mast cells, or combinations thereof.

[0023] In some embodiments, the phagocytes include macrophages. In some embodiments, the phagocytes include immune cells. In some embodiments, the phagocytes include tumor cells and immune cells.

[0024] Particles

[0025] The methods and systems of the present disclosure may be utilized to evaluate the phagocytosis of various particles by phagocytes. For instance, in some embodiments, the particles include, without limitation, phagocyte target cells (either prokaryotes or eukaryotes), cellular debris, pathogens, dead cells, apoptotic cells, bacteria, fungi, viruses, parasites, beads, or combinations thereof. In some embodiments, the particles may be pretreated with antibodies.

[0026] Obtaining phagocytes and / or particles

[0027] In some embodiments, the methods of the present disclosure also include a step of obtaining the phagocytes and / or particles. In some embodiments, the phagocytes and / or particles are obtained from a tissue or a blood sample. In some embodiments, the phagocytes and / or particles are obtained by methods that include, without limitation, flow cytometry, positive flow sorting, negative flow sorting, magnetic sorting, or combinations thereof.

[0028] SurfacesPCT Application Attorney Docket No. AF23853.P209WOUH ID No. 2024-089

[0029] The methods of the present disclosure may be utilized to evaluate the phagocytosis of phagocytes on various surfaces. Additionally, the systems of the present disclosure may include various surfaces. For instance, in some embodiments, the surfaces include a well. In some embodiments, the well includes a nanowell. In some embodiments, the well includes an array of individual wells. In some embodiments, the individual wells include individual nanowells. In some embodiments, phagocytes and particles are positioned in each of the wells for simultaneous evaluation of multiple phagocytoses in different wells.

[0030] Visualization of phagocytosis

[0031] The methods and systems of the present disclosure may be utilized to visualize the phagocytosis of particles by phagocytes in various manners. For instance, in some embodiments, the visualization occurs at a single-cell level. In some embodiments, the visualization occurs in real-time. In some embodiments, the visualization occurs at sequential intervals for a period of time. In some embodiments, the period of time ranges from about 1 hour to about 24 hours. In some embodiments, the sequential intervals range from about 1 minute to about 10 minutes.

[0032] In some embodiments, phagocytosis is visualized using pHRodo, lysosomal dyes or other pH sensitive dyes. In some embodiments, phagocytosis is visualized using phase contrast or brightfield microscopy.

[0033] In some embodiments, the visualization of the phagocytosis of particles by phagocytes includes imaging the phagocytosis. In some embodiments, the imaging includes timelapse imaging. In some embodiments, the imaging includes microscopy.

[0034] In some embodiments, the visualization of the phagocytosis of particles by phagocytes includes labeling the phagocytes. In some embodiments, the phagocytes are labeled by staining with fluorescent-based detection reagents. In some embodiments, the phagocytes are labeled using dyes. In some embodiments, the dyes include, without limitation, DAPI, PKH family of dyes, CellTrace family of dyes, or combinations thereof. In some embodiments, targets are labeled using dyes or fluorescent antibodies.PCT Application Attorney Docket No. AF23853.P209WOUH ID No. 2024-089

[0035] In some embodiments, the visualization of the phagocytosis of particles by phagocytes includes the use of a sensor associated with the surface (e.g., sensor 25 in FIG. IB). In some embodiments, the sensor is in the form of a bead. In some embodiments, the bead includes diameters that range from about 1 pm to about 10 pm. In some embodiments, the bead includes diameters that range from about 3 pm to about 5 pm.

[0036] In some embodiments, the sensor includes an analyte binding agent. In some embodiments, the analyte binding agent includes, without limitation, genes, nucleotide sequences, interference RNA (RNAi), antisense oligonucleotides, peptides, antisense peptides, antigene peptide nucleic acids (PNA), proteins, antibodies, or combinations thereof.

[0037] In some embodiments, the analyte binding agent is directed against an analyte of interest. In some embodiments, the analyte of interest includes, without limitation, secreted proteins, cell lysate components, cellular receptors, metabolites, lipids, microvesicles, exosomes, microparticles, small molecules, protein-carbohydrates, or combinations thereof.

[0038] In some embodiments, the analyte of interest is captured by the sensors. In some embodiments, the analyte of interest is subsequently characterized. In some embodiments, the analyte of interest is characterized by methods that include, without limitation, mass spectrometry, sequencing, microscopy, nucleic acid hybridization, immunoassay-based detection, or combinations thereof. In some embodiments, the phagocytes are lysed prior to incubation with the sensors.

[0039] In some embodiments, the visualization of the phagocytosis of particles by phagocytes includes performing Time-lapse Imaging Microscopy in Nanowell Grids (TIMING) assay on the surface. In some embodiments, the TIMING assay includes: (1) auto-localizing a surface containing the phagocytes by the use of image analysis algorithms to obtain an image of the surface; and (2) performing automated cell segmentation of phagocytes. In some embodiments, the automated cell segmentation of phagocytes occurs by methods that include, without limitation, confinement- constrained cell segmentation of phagocytes, confinement-constrained cell tracking of phagocytes or combinations thereof.

[0040] In some embodiments, the TIMING assay also includes: (1) characterizing the molecular profiles of the phagocytes; and (2) integrating the TIMING data and the molecular profiles data of the phagocytes. In some embodiments, a sensor is utilized as a marker to enable auto-focusing of the phagocytes during a TIMING assay.PCT Application Attorney Docket No. AF23853.P209WOUH ID No. 2024-089

[0041] In some embodiments, the processors of the present disclosure include programming instructions for implementing such TIMING assays. For instance, in some embodiments, the processors of the present disclosure include programming instructions for: (1) auto-localizing a surface containing the phagocytes by the use of image analysis algorithms to obtain an image of the surface; and (2) performing automated cell segmentation of the phagocytes. In some embodiments, the processor further includes programming instructions for: characterizing the molecular profiles of the phagocytes; and integrating the auto-localizing and cell segmentation data of the phagocytes with the molecular profiles data of the phagocytes. In some embodiments, the processor further includes programming instructions for: characterizing one or more molecular profiles of the phagocytes; and integrating the visualized phagocytosis and the molecular profiles of the phagocytes.

[0042] Characterizing molecular profiles of phagocytes

[0043] The methods and systems of the present disclosure may be utilized to characterize various molecular profiles of phagocytes. For instance, in some embodiments, the characterized molecular profiles include, without limitation, transcription activity, transcriptomic profile, gene expression activity, genomic profile, protein expression activity, protein secretion activity, proteomic profile, protein interaction activity, cellular receptor expression activity, lipid profile, lipid activity, carbohydrate profile, microvesicle activity, glucose activity, metabolic profile, or combinations thereof.

[0044] The characterization of the molecular profiles of phagocytes can occur in various manners. For instance, in some embodiments, the characterization occurs at a single-cell level. In some embodiments, the characterization occurs by methods that include, without limitation, DNA analysis, DNA sequencing, RNA analysis, RNA sequencing, protein analysis, protein sequencing, lipid analysis, metabolite analysis, mass spectrometry, or combinations thereof.

[0045] In some embodiments, the characterization of the molecular profiles of phagocytes occurs by RNA or DNA sequencing. In some embodiments, the RNA or DNA sequencing occurs by methods that include, without limitation, whole transcriptome analysis, whole genome analysis, barcoded sequencing of whole or targeted regions of the genome, or combinations thereof.

[0046] In some embodiments, the characterization of the molecular profiles of phagocytes occurs by protein analysis. In some embodiments, the protein analysis occurs at the proteomic level by multiplexed fluorescent staining.PCT Application Attorney Docket No. AF23853.P209WOUH ID No. 2024-089

[0047] Integration of phagocytosis and molecular profiles

[0048] Various methods may be utilized to integrate visualized phagocytosis and molecular profiles of phagocytes. For instance, in some embodiments, the integration occurs at a single-cell level. In some embodiments, the integration includes correlating phagocytosis of the particles by the phagocytes to gene expression or transcription activities of the phagocytes. In some embodiments, the integration includes correlating the phagocytosis of the particles by the phagocytes to protein secretion activity of the phagocytes. In some embodiments, the integration includes correlating cellular interaction activities of the phagocytes and particles to protein expression activity of the phagocytes.

[0049] Applications

[0050] The methods and systems of the present disclosure can have various applications. For instance, in some embodiments, the methods and systems of the present disclosure may be utilized for at least one of predicting clinical outcome of a treatment, screening cells, retrieving cells, facilitating a treatment, diagnosing a disease, visualization of cellular activity, or combinations thereof.

[0051] In some embodiments, the methods and systems of the present disclosure may be utilized to facilitate a treatment. In some embodiments, the treatment includes immunotherapy.

[0052] In some embodiments, the methods and systems of the present disclosure may be utilized to monitor cellular activity. In some embodiments, the cellular activity includes an immune response.

[0053] In some embodiments, the methods and systems of the present disclosure may be utilized for screening of cells. In some embodiments, the cells include giant multinucleated cells. In some embodiments, the cells include multi-phagocytosing cells.

[0054] Implementation of a treatment decision

[0055] In some embodiments, the methods of the present disclosure also include a step of implementing a treatment decision based on the evaluation of phagocytosis. In some embodiments, the systems of the present disclosure include programming instructions for recommending a treatment decision based on the evaluation. In some embodiments, the treatment decision includes immunotherapy. In some embodiments, the immunotherapy may include isolation of monocytes from a subject (e.g., a human) and performing phagocytosis assays against antibody coated target cells.

[0056] Additional embodimentsPCT Application Attorney Docket No. AF23853.P209WOUH ID No. 2024-089

[0057] Reference will now be made to more specific embodiments of the present disclosure and experimental results that provide support for such embodiments. However, Applicant notes that the disclosure below is for illustrative purposes only and is not intended to limit the scope of the claimed subject matter in any way.

[0058] Example 1. Single-cell profiling of phagocytosis using microscopy

[0059] Applicant has developed methods of evaluating and monitoring phagocytosis by placing a cell population on an area to assay their phagocytic behavior at the single cell level as a function of time. The method allows for identifying cell(s) of interest based on their phagocytic behavior; characterizing the molecular profile of the cell(s); and correlating the obtained information. Sensors associated with the area may be utilized. The molecular profiles of the cell(s) can be characterized by various methods, such as DNA analysis, RNA analysis, and protein analysis. The phagocytic behavior and molecular profiles can then be correlated and be used to predict clinical outcomes of a treatment, screening cells, facilitating a treatment and diagnosing a disease.

[0060] In particular, the developed method can help one better understand the underlying mechanism that control how cells interact with and phagocytose targets at the single-cell level in vitro and correlate the behavior with molecular profiles of the cells. This in turn allows one to better predict outcomes to treatments and develop better treatments for diseases.

[0061] To the best of Applicant’s knowledge, prior studies did not develop a method that can assay phagocytosis at the single cell level in a high throughput manner and correlate the observed behaviors to the molecular profiles of the cell.

[0062] Example 1.1. Experiment protocols for phagocytosis of COVID- 19 Spike Proteins by THP-1 cellsPCT Application Attorney Docket No. AF23853.P209WOUH ID No. 2024-089

[0063] The in-house fabrication of the polydimethylsiloxane (PDMS) nanowell array was previously described (Liadi et al., Cancer Immunol Res. 2015 May;3(5):473-82. doi: 10.1158 / 2326-6066). The chip was placed in a plasma chamber (Harrick Plasma Inc; Ithaca, NY) for two minutes and treated with PLL

[0020] -g[3.5]- PEG(2) / PEG(3.4)- biotin(50%) (SuSoS: Zurich, Switzerland) for assays using THP-1 cells and 1 pm polystyrene florescent beads. The beads were resuspended in phosphate- buffered saline (PBS) before being incubated with COVID- 19 Spike Protein (HexapPro) for 1 hr before being washed with PBS. Then, pHrodo Red(ThermoFisher Scientific) was conjugated to FBS and added to the beads. The mixture was then allowed to incubate for 1 hour before being washed and reconstituted in 5% FBS in PBS. The THP-1 cells were resuspended at a density of 1 million cells / mL in complete RPMI media and loaded onto the nanowell array. The beads were similarly reconstituted at a density of 10 million / mL in complete media and loaded onto the nanowell array. The array was then resuspended in IMDM with 10% FBS and imaged using a Zeiss Axio Observer (Oberkochen, Germany) in the Brightfield, Alexa 488 and TexasRed channels over 73 time points for 6 hrs. The results are shown in FIG. 2.

[0064] Example 1.2, Experiment protocols for phagocytosis of B16 cells by THP-1 cells

[0065] The in-house fabrication of the PDMS nanowell array was previously described (Liadi et al., Cancer Immunol Res. 2015 May;3(5):473-82. doi: 10.1158 / 2326-6066). The chip was placed in a plasma chamber (Harrick Plasma Inc; Ithaca, NY) for two minutes and treated with PLL

[0020] -g[3.5]- PEG(2) / PEG(3.4)-biotin(50%) (SuSoS; Zurich, Switzerland) for assays using THP-1 cell derived macrophages and human Her2 expressing B16 cells. THP-1 cells were stimulated for 72hrs using 150nM PMA (InvivoGen). THP-1 cells were washed with PBS before being labelled using PKH26 Red (Sigma- Aldrich). The cells were then washed 3 times and reconstituted in complete RPMI at a density of 1 million cells / mL. The B 16 HER2+ cells were reconstituted in complete media at 1 million cells / mL and incubated with 2.5 pg / mL Trastuzumab for Ihr. Both cells were then loaded onto the nanowell array and resuspended in IMDM with 10% FBS and imaged using a Zeiss Axio Observer (Oberkochen, Germany) in the Brightfield and TexasRed channels over 73 time points for 6 hrs. The results are shown in FIG. 3.PCT Application Attorney Docket No. AF23853.P209WOUH ID No. 2024-089

[0066] Without further elaboration, it is believed that one skilled in the art can, using the description herein, utilize the present disclosure to its fullest extent. The embodiments described herein are to be construed as illustrative and not as constraining the remainder of the disclosure in any way whatsoever. While the embodiments have been shown and described, many variations and modifications thereof can be made by one skilled in the art without departing from the spirit and teachings of the invention. Accordingly, the scope of protection is not limited by the description set out above, but is only limited by the claims, including all equivalents of the subject matter of the claims. The disclosures of all patents, patent applications and publications cited herein are hereby incorporated herein by reference, to the extent that they provide procedural or other details consistent with and supplementary to those set forth herein.

Claims

PCT Application Attorney Docket No. AF23853.P209WOUH ID No. 2024-089CLAIMS1. A method of evaluating phagocytosis at a single-cell level, said method comprising: placing one or more phagocytes and one or more particles on a surface; and visualizing the phagocytosis of the particles by the phagocytes at a single-cell level and in real-time.

2. The method of claim 1, wherein the phagocytes are selected from the group consisting of immune cells, T cells, B cells, monocytes, macrophages, neutrophils, dendritic cells, natural killer cells, fibroblasts, stromal cells, stem cells, progenitor cells, tumor cells, tumor stem cells, tumor infiltrating lymphocytes, macrophages, mast cells, or combinations thereof.

3. The method of claim 1 , wherein the phagocytes comprise macrophages.

4. The method of claim 1, wherein the phagocytes comprise tumor cells and immune cells.

5. The method of claim 1, wherein the particles are selected from the group consisting of phagocyte target cells, cellular debris, pathogens, dead cells, apoptotic cells, bacteria, fungi, viruses, parasites, beads, or combinations thereof.

6. The method of claim 1, further comprising a step of obtaining the phagocytes, particles, or combinations thereof.

7. The method of claim 1, wherein the surface comprises a well.

8. The method of claim 7, wherein well comprises a nanowell.

9. The method of claim 7, wherein the well comprises an array of individual wells.PCT Application Attorney Docket No. AF23853.P209WOUH ID No. 2024-08910. The method of claim 9, wherein phagocytes and particles are positioned in each of the wells for simultaneous evaluation of multiple phagocytoses in different wells.

11. The method of claim 1, wherein the visualizing occurs at sequential intervals for a period of time.

12. The method of claim 1, wherein the visualizing comprises imaging the phagocytosis, and wherein the imaging comprises timelapse imaging.

13. The method of claim 1, wherein the visualizing comprises labeling the phagocytes.

14. The method of claim 1, wherein the visualizing comprises the use of a sensor associated with the surface.

15. The method of claim 14, wherein the sensor comprises an analyte binding agent, wherein the analyte binding agent is directed against an analyte of interest, and wherein the analyte of interest is selected from the group consisting of secreted proteins, cell lysate components, cellular receptors, metabolites, lipids, microvesicles, exosomes, microparticles, small molecules, protein-carbohydrates, or combinations thereof.

16. The method of claim 15, wherein the analyte of interest is captured by the sensors, and wherein the analyte of interest is subsequently characterized.

17. The method of claim 1, wherein the visualizing comprises performing Time-lapse Imaging Microscopy in Nanowell Grids (TIMING) assay on the surface, wherein the TIMING assay comprises: auto-localizing a surface containing the phagocytes by the use of an image analysis algorithm to obtain an image of the surface; andPCT Application Attorney Docket No. AF23853.P209WOUH ID No. 2024-089 performing automated cell segmentation of phagocytes.

18. The method of claim 17, wherein the TIMING assay further comprises: characterizing the molecular profiles of the phagocytes; and integrating the TIMING data and the molecular profiles data of the phagocytes.

19. The method of claim 17, wherein a sensor is utilized as a marker to enable auto-focusing of the phagocytes during a TIMING assay.

20. The method of claim 1, further comprising steps of: characterizing one or more molecular profiles of the phagocytes; and integrating the visualized phagocytosis and the molecular profiles of the phagocytes.

21. The method of claim 20, wherein the characterized molecular profiles are selected from the group consisting of transcription activity, transcriptomic profile, gene expression activity, genomic profile, protein expression activity, protein secretion activity, proteomic profile, protein interaction activity, cellular receptor expression activity, lipid profile, lipid activity, carbohydrate profile, microvesicle activity, glucose activity, metabolic profile, or combinations thereof.

22. The method of claim 20, wherein the characterizing and integrating occur at a single-cell level.

23. The method of claim 22, wherein the integrating comprises correlating the phagocytosis of the particles by the phagocytes to protein secretion activity of the phagocytes, correlating cellular interaction activities of the phagocytes and particles to protein expression activity of the phagocytes, or combinations thereof.PCT Application Attorney Docket No. AF23853.P209WOUH ID No. 2024-08924. The method of claim 1. further comprising a step of implementing a treatment decision based on the evaluation.

25. The method of claim 24, wherein the treatment comprises immunotherapy.

26. A system for evaluating phagocytosis of a particle by a phagocyte at a single-cell level, wherein the system comprises a non-transitory processor operable for evaluating visualized phagocytosis of the particle by the phagocyte.

27. The system of claim 26, wherein the system further comprises a sensor operable for visualizing the phagocytosis of the particle by the phagocyte.

28. The system of claim 26, wherein the processor comprises programming instructions for integrating the visualized phagocytosis and molecular profiles of phagocytes.

29. The system of claim 26, wherein the processor further comprises programming instructions for recommending a treatment decision based on the evaluation.

30. The system of claim 26, wherein the system further comprises a surface operable for receiving the phagocyte and the particle.

31. The system of claim 30, wherein the surface comprises a well.

32. The system of claim 31, wherein well comprises a nanowell.

33. The system of claim 31, wherein the well comprises an array of individual wells.

34. The system of claim 26, wherein the processor comprises programming instructions for:PCT Application Attorney Docket No. AF23853.P209WOUH ID No. 2024-089 auto-localizing a surface containing the phagocytes by the use of an image analysis algorithm to obtain an image of the surface; and performing automated cell segmentation of the phagocytes.

35. The system of claim 34, wherein the processor further comprises programming instructions for: characterizing the molecular profiles of the phagocytes; and integrating the auto-localizing and cell segmentation data of the phagocytes with the molecular profiles data of the phagocytes.

36. The system of claim 26, wherein the processor further comprises programming instructions for: characterizing one or more molecular profiles of the phagocytes; and integrating the visualized phagocytosis and the molecular profiles of the phagocytes.

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