Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments

1. Chip design for adherent and floating cells 2D co-cultures

NOTE: The 2D co-culture layout (Figure 1A-C) is characterized by three chambers (100 µm high) interconnected by two sets of microchannel arrays (500 x 12 x 10 µm³, L×W×H). The intermediate chamber forms two closed dead-end compartments which block floating immune cells overflowing into the tumor site during the loading step 2.5. This device type is useful for real-time bidimensional measurements of single-cell (either adherent or floating) motility, and of cell-cell interactions^{16,27,28,30,31}. A typical cell migration study (conducted from several hours to several days) combines live-cell microscopy with image-processing algorithms⁴⁵, in order to translate the acquired image sequences into numerical features²⁵. Based on the migratory patterns, several biophysical indicators can be estimated, such as the displacement and velocity of cells, as well as the duration of immune cell and target cell interactions²⁴.

1. Preparation of cancer and PBMC cells
   1. Cancer cell culture
      NOTE: MDA-MB-231 triple-negative [estrogen receptor (ER)-, progesterone receptor (PR)-, and human epidermal growth factor receptor 2 (HER2)-] human breast adenocarcinoma cells are routinely grown in a Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS), 2 mM L-glutamine, 100 IU mL⁻¹ penicillin G sodium salt and 100 µg mL⁻¹ streptomycin sulfate (growth medium), under standard culture conditions (37 °C and 5% CO₂).
      1. To optimize cell culture growth, plate MDA-MB-231 cells in 75 cm² flasks at a density of 1 × 10⁶ cells mL^-1 in 12-to-15 mL of growth medium.
      2. When cells reach the 75-80% of confluence, discard the growth medium, wash cells with pre-warmed phosphate-buffered saline (PBS) to completely remove FBS, and then detach them with pre-warmed trypsin (1-to-2 min at 37 °C).
      3. Add growth medium to inactivate trypsin enzymatic activity and collect detached cells. Wash cells twice for 5 min at 1,100 x g at room temperature (RT).
      4. Count cells in a cell counting slide by means of Trypan Blue dye exclusion test and then reseed them either for maintenance culture (for no more than 6 passages from thawing) or for experimental procedures.
      5. For microfluidic experiments, seed 1 × 10⁶ cells in 6-well plates in 3 mL of growth medium and either treat with 25 µM doxorubicin (DOXO) or an equal volume of DOXO solvent (PBS) as control.
      6. Four-to-6 hours after, wash DOXO-treated cells twice with pre-warmed PBS for 5 min at 1,100 x g at RT.
      7. Count DOXO-treated and PBS-treated control cells as above (see step 1.1.1.5) and set the co-culture with peripheral blood mononuclear cells (PBMCs) in microfluidic devices.
   2. PBMC isolation
      1. Collect venous whole blood (10 mL approximately) from healthy volunteers in heparinized vials and gently mix by inverting the tube 2-to-4 times⁴⁷.
      2. Dilute blood 1:1 with PBS and layer over 10 mL of density gradient medium Lymphoprep in a 50 mL tube.
         ​NOTE: Ensure to do the layering very gently and slowly to let blood and Lymphoprep form two distinct layers.
      3. Centrifuge tubes for 30 minutes at 400 x g at 4 °C in a swing-out bucket without brakes. Four distinct layers will form: (i) plasma at the top, (ii) a white and cloudy layer containing PBMCs, (iii) Lymphoprep, and (iv) a pellet of erythrocytes and granulocytes.
      4. Aspirate gently PBMCs with a 2 mL pipette and immediately resuspend in warm growth medium (same used in step 1.1.1) and wash twice for 5 min at 1,100 x g at RT.
      5. Count pelleted PBMCs as above (see step 1.1.1.5) and either use for experimental procedures or freeze for long-term storage.
2. Plating the cells in 2D chips
   NOTE: PBMCs are not stained in this protocol. To characterize specific phenotypes on-chip, immune cell sub-populations can be isolated by immunomagnetic bead selection, stained with fluorescent cell trackers, re-mixed with the unlabeled remaining fraction, and thus confronted with target cancer cells, as reported in the on-chip experiments, described in Vacchelli et al.²⁷, and in Racioppi et al.³⁴.
   1. Before starting co-culture experiments and to facilitate the addition of reagents, activate stored chips by an oxygen plasma treatment for a few seconds. Immediately fill reservoirs with deionized water or PBS to keep PDMS (polydimethylsiloxane) surfaces hydrophilic until plating steps.
      NOTE: PDMS is intrinsically hydrophobic, which may result in difficulties in operating and in the entrapment of air bubbles in microchannels. See step 7 in the supplementary file providing details about oxygen plasma activation.
   2. Sterilize under a UV cabinet for 20 min, wash 2-3 times with fresh PBS, and then incubate with culture media for 1 h. Keep chips in incubator until performing plating steps.
   3. Withdraw excess media from all six reservoirs. Take care to avoid sucking up media from the main culture chambers.
   4. Slowly apply 1 × 10⁵ cancer cells resuspended in 10-20 µL of growth medium in the upper left-hand reservoir, and then in the lower well (Figure 1A, reservoirs 1 and 2). Wait 5 min to let cells adhere into the tumor chamber. Some cells will settle and attach in the reservoirs.
      NOTE: Insert cellular suspension next to the channel openings. This procedure is applied to MDA-MB-231 cancer cells, other lines will require cell density optimization. To improve cancer cell attachment, a coating functionalization of surfaces (e.g., poly-L-lysine, fibronectin) can be performed. Please, refer to previously published protocols for coating steps^{16, 48,49,50}.
   5. On the right side gently pipet 1 × 10⁶ PBMCs resuspended in 50 µL of growth medium into wells 3 and 4 (see Figure 1A, reservoirs 3 and 4).
      NOTE: After flowing, PBMC will distribute into the intermediate chamber creating a "front", which represents the starting point of the experiment.
   6. Fill all the six reservoirs with up to 100-150 µL of growth medium. Under a microscope check that cells have distributed correctly in the culture compartments as depicted in Figure 1D-E. Final volumes can vary with the size of the reservoirs. Adjust volumes to be equal in all wells.
   7. Place the chips back in the incubator for approximately 1 h to stabilize the system prior to time-lapse recording. Add fresh medium every 3 days, as it may be subjected to evaporation losses.
      ​NOTE: The system is compatible with both live/dead-cell analysis and dynamic multiplex cytokine secretion profiling from conditioned media. For chemokine analyses, up to 200-250 µL aliquots of supernatants may be accessible by collecting media from the two reservoirs of each compartment. Classical ELISA and Luminex cytokine profiling assays require about 50 µL of supernatants. Please see ^{51, 52} examples of studies of other labs performing cytokine profiling on OOC models. 
3. Time-lapse acquisition of unlabeled cancer and immune cells 
   NOTE: Typically, 3 chips are arranged on a single microscope slide (see Figure 1A for 2D chip and Figure 4B for 3D chip). Using stage holders allocating 4 slides, co-cultures can be suitable to be monitored by automated high-content microscopy to analyze large batches of experimental conditions. Chips can be easily mounted on slides with thickness equal to 1 mm or 170 microns (plastic or glass coverslips, 6-well optical bottom multi-wells) for high-resolution confocal imaging.
   1. Record bright-field image series of unlabelled cells by means of a video microscopy setup equipped with an incubation system.
      NOTE: Here time-series datasets (time window: 48 h, frame rate: 2 min) were acquired with a fluorescence microscope, equipped with a 4x objective and CMOS 1.3M pixels, optimized to fit into a standard cell culture incubator.
   2. Warm up the microscope for at least 2 h to equilibrate to 37 °C and 5% CO₂ before starting acquisition.
   3. Select the window of observation by centering the microchannel array between the tumor and the central compartment. This allows to visualize the dynamics of immune infiltration and the interactions within the region in which cancer cells are seeded.
   4. Adjust the illumination intensity and focus of cancer and immune cells.
   5. For launching time-lapse acquisition, optimize frame rate and time duration according to experiment and cell type under study.
      NOTE: Imaging conditions must be optimized to avoid excessive photo-exposure while maintaining a good signal-to-noise ratio (SNR). As immune cells are very motile, the acquisition frame rate needs to be sufficiently high to follow the dynamic process of interest and enable easy tracking⁵³. A compromise should be reached between the tracking algorithm, the compatibility with the size of the resulting dataset, and the viability, density, and motility of observed cells.
   6. At the end of the time-lapse, use the function Import Image Sequence and Save as of the ImageJ software to convert the frame dataset in a 25 fps uncompressed video file.
      NOTE: The generated video file is now ready for cell tracking analysis. Here, RGB (1280×1024 pixels) images were collected with a spatial resolution of 1.33 µm/pixel. A 24 h duration movie (3.5 GB stack) of a single field of view (FOV) consists of 720 frames for each condition. 
4. Data analysis: Semi-automatic extraction of unlabeled immune tracks by Trackmate
   NOTE: Here, immune motility analysis in 2D unlabeled time-lapse images is carried on using TrackMate⁵⁴, an open-source toolbox available in the Fiji/ImageJ software bundle (https://imagej.nih.gov/ij/). Several algorithms are provided to perform automated single-particle tracking⁵⁵ (SPT) of spot-like structures. They have been applied efficiently to fluorescent images, where objects are bright over a dark background with high SNR (i.e., sub-resolution fluorescent spots, labeled traffic vesicles, nuclei)^{1,25,56,57,58}. SPT is mainly based on two sequential steps. First, objects are localized with identified positions in multiple frames (segmentation), as schematized in Figure 2. In the second stage (particle linking), detected spots are linked over consecutive frames to estimate motion and reconstruct their trajectories, in the shape of a track (Figure 3). Numerical features can be computed from each extracted X, Y, Z coordinates array over time. Extended documentation is reported in ⁵⁴ as well as online (http://imagej.net/TrackMate), following the Getting started with TrackMate tutorial. The accuracy of the process can be inspected immediately, handling an intuitive graphical user interface (wizard-like GUI) that enables users, at every step, to readjust settings. The following part briefly depicts how to use Trackmate for image processing and quantification steps, applied to visible light images:
   1. Drag and drop the full-time video/image stack on the Fiji toolbar.
   2. Calibration stack setup (Figure 2A).
      1. Check the dimensionality and assign the image properties by selecting Image> Properties. Fill Unit of length, Pixel dimensions and Frame interval boxes.
         NOTE: To perform the calibration, use the known length of the microchannels (500 µm, Figure 1C) and divide by the corresponding measured length in pixels. For 2D time-series, make sure to swap Z/T field entering 1 as z-slice and the correct number of movie frames. If not accomplished, Trackmate quantitative outputs and parameters will be reported in pixel units and timeframes.
   3. Pre-processing of images.
      1. To enhance the correct discrimination of immune cells from a noisy background, pre-process bright-field images to compensate artifacts. Ensure that datasets consist of 8-bit TIFF images (brightness range: 0-255).
         NOTE: Uneven illumination, low SNR, and contamination by small debris particle in visible light images could compromise the success of a cell tracking process. Here, time-series datasets are pre-processed through background subtraction, brightness/contrast adjust function, and by local image subtraction of a Gaussian blur from original images. There are other different analysis toolkits available in ImageJ for processing and segmentation of phase-contrast or bright-field images, including Empirical Gradient Threshold (EGT)⁵⁹.
   4. First calibration panel (Figure 2A)
      1. With image stack selected, start Trackmate (Plugins>Tracking).  Revise/confirm the dimensionality and temporal window of data (i.e., pixel-width and frame interval).
         NOTE: TrackMate automatically reads in the image properties box to give the final tracking results in calibrated physical units (i.e., µm and minutes).
      2. Define a region of interest to compute the extraction of immune tracks, by manually inserting values or by drawing a closed area over the active image, and then pressing the Refresh source button. To extract global immune migration paths, select rectangular regions respectively on the right side of microchannels (central chamber, Figure 1E) and on the left (tumor chamber, Figure 1D). To analyze interactions between cancer and immune hotspots, draw circular sub-regions by using ROI tools (go to Edit → Selection → Specify).
         NOTE: When running for the first time this toolbox on a new biological application, spend the necessary time to optimize settings for reconstructing the tracks.
         NOTE: Perform manual tracking of the cell trajectories (about 50-100 cells) to find empirically the right configuration and next to validate as a benchmark the reliability of automatic extraction of movements. Additionally, work initially on a smaller area to easily check the accuracy of the chosen parameters.
   5. Immune spots detection step (Figure 2B)
      1. Select the default Laplacian of Gaussian (LoG) detector. The LoG detector works to find bright, blob-like, roundish objects and applying a Laplacian of Gaussian filter on the image tuned for intermediate spot sizes (5-to-20 pixels in diameter).
      2. In Estimated Blob Diameter (here, 10-13 µm) enter a value slightly bigger than the expected spot size. Increase Threshold (here 1-3 µm) value until extra spurious background spots are reduced possibly without removing the object features. Detections below Threshold value (based on a quality metrics) will be discarded from subsequent analysis. Check the box for the median filter and sub-pixel localization to improve the quality of spot detection.
      3. Use the Önizleme button to view and quickly inspect identified immune cells overlaid on the images by magenta-colored circles.
         NOTE: Mistakes during the detection will have a considerable impact on the linking process. Other unwanted detections can be corrected in the subsequent menus by user-defined filters (i.e., by spot intensity, size, or position).
   6. Once satisfied with selections, hit Next.
      NOTE: These settings may vary depending on experimental setup and acquisition imaging modalities (e.g., FOV, objective magnification, bright-field or fluorescent images), cell type (adherent or floating cells), from slow or fast motility, kind of cellular behavior (interacting or not) and low/medium/high density in the observation area.
   7. Proceed and skip Initial Thresholding menu. Select the Hyperstack Displayer window.
   8. Set filters on spots panel (Figure 2C).
      1. Select: Uniform Color. Filters, as shown in Figure 2C, can be added to retain labeled spots with feature values, displayed in a histogram, above or below a reversible threshold.
   9. Tracker selection stage (Figure 3A). Choose the Simple LAP tracker, as particle linking algorithm asking for three fields to fill (in this case, "Linking max distance": 30-50 µm, "Gap-closing max distance": 25-50 µm, "Gap-closing max frame gap": 4-6). This detector manages gap-closing events, with cost linking calculation solely based on their respective distance.
      NOTE: The maximal allowed linking distance limits the spatial search range for candidate matching spots, corresponding to the maximally allowed displacement traveled by between two subsequent frames (Figure 3D).
      1. Provide larger values of maximal displacement when the fragmentation of tracks of highly motile particles is noticed.
         NOTE: Two links will not be connected if the frame-to-frame movement is larger than the given maximal distance value. If segments bridge badly two different cells, decrease the value of maximal displacement.
      2. Try to reconnect missing spots, varying the values of "the max distance for gap closing" and "the maximal frame gap".
         NOTE: These parameters deal with gap-closing events in non-adjacent frames. Spot disappearance may occur for some frames (i.e., out of focus particles, cells leaving out and in the FOV, segmentation failures in a noisy image).
         NOTE: To handle splitting or merging events, opt for LAP linker as detector which introduces linking cost matrix penalties.
   10. Click Next to run the tracking computation. Press Next.
   11. Filtering tracks panel (Figure 3B). Change the color of immune paths selecting, from the drop-down menu, "Track ID" or other track features. At this point, choose optionally to set interactive filters functional, to improve the quality of the outcome and revisit the procedure.
       NOTE: Spurious spots arise from noise in the image and loss of feature quality. This will generate short segments while cells of interest can be tracked over many frames.
       1. To remove short paths, try to filter out, based on the number of spots they contain. Additionally, sort tracks using a combination of options such as Track displacement, Track duration or Minimal/Mean/MaximalVelocity to exclude false or unwanted tracks (with fewer frames respect to overall duration of time-lapse or involving dirty or not moving particles) from further post-processing.
          NOTE: The choice of filters can vary depending on the specific application and biological system.
   12. Examine all tracks in the Display Options interface, scroll through time, and verify how accurate tracks match cell migration paths. The drop-down menu provides color codes for spots and paths for easy visualization and filtering by several modalities (e.g., kinetic parameters, intensity, temporal or spatial position).
       NOTE: For tracking high-density cultures, or high-motile cells, increase the acquisition frame rate minimizing cell displacement traveled in consecutive time intervals.
   13. Manual correction of segmentation and linking mistakes (Figure 3E).
       1. To enhance further the quality of results, edit manually spots (debris particles, stationary cells) and remove erroneous tracks deriving from detected tumor boundaries when analyzing tumor-immune interaction ROIs.
       2. First, select the TrackMate tool in the ImageJ toolbar. For eliminating an existing spot throughout the whole stack, press shift and create by mouse cursor a ROI mask over the target spot (edited in a green circle), and then hit the DEL key.
       3. For adding a new spot (in case of missing tracks due to spots disappearance) press the A key, laying the mouse at the pointed location. Repeat the tracks-linking computation process after this step.
   14. When satisfied, select Analysis in the Display Options panel to generate three text files (Figure 3C and 3F). The table in "Spots in tracks statistics" provides the spatiotemporal coordinates of immune spots (X-Y-Z positions of the cells labeled with the associated frame and track number). "Links in tracks statistics" and "Track statistics" contain information relative to the tracks: track durations, number of detected gaps or spots, track initial and stop-frame, etc. Save and export for each dataset.
       NOTE: When clicking on a row within the result windows, the respective spot, link, or track is activated within the time-lapse video for visual inspection. Repeat the filtering steps to select/remove tracks. All future exported data will be updated. TIP: Track initial and stop-frame and track duration values can be exploited to calculate times of contact between cancer and immune cells when processing ROIs of interaction.
   15. Press the Save button to generate a resulting XML file containing all the parameter values, the path to images, and spot positions in time. The ''Load TrackMate file'' command (Plugins> Tracking) restores the whole process session for each movie file individually.
   16. Move to the last panel of the GUI called Select an action. In the list, use Captureoverlay > Execute function to produce a video with tracks overlaid. TIP: "Plot N-spots vs time" option may be used to compute the spatial density of immune cells in a ROI (Figure 6B, right panel).
   17. Post-processing analysis and migration statistics
       1. Analyze the raw positional data directly in Trackmate or export data to calculate comprehensive kinetic parameters²⁹ (i.e., total trajectory length, Euclidean distance, confinement ratio, mean-squared displacement⁵⁶, average or instantaneous track velocity, arrest coefficient, distribution of angles of migration, Forward migration index, mean straight-line speed) to classify immune cell migration behavior (e.g., directed or diffusive motion^30,31) and response to target cancer cells (e.g., treated vs control).
          ​NOTE: Additional useful plugins such as The Chemotaxis and Migration Tool (http://ibidi.com/software/chemotaxis_and_migration_tool/) provides various graphs (e.g., Rose or sector plots, such as depicted in Figure 6) and statistical tests for advanced analysis and visualization of experimental migration and chemotaxis data. Combining cell tracking and cell segmentation algorithms^24,25,45 may enable measurements of morphological metrics at the single-cell level (i.e., cell surface area, the major and minor axis length, and the cell aspect ratio).

2. 3D immuno-competent cancer on-chip model in a competitive assay

NOTE: The 3D chip design, depicted in Figure 4, consists of 5 major compartments: a central one for the floating immune cells intake, two side regions for embedding tumor cells in hydrogel matrices (150-250 µm high), and media perfusion chambers. Immune and tumor chambers are connected by two sets of narrow arrays of microchannels (200×12×10 µm³, L×W×H, Figure 4E). Regularly 100 µm-spaced trapezoidal isosceles micropillars (about 25-30 interfaces for each side gel region, Figure 4C) work as barriers to confine gel solution during injection exploiting the balance between surface tension and capillary forces^60,61 and connect tumor regions to the two lateral additional media chambers in order to set a gel-liquid interface (Figure 5). The detailed features of the 3D competitive assay are shown in Figure 4. Preferential migration of immune cells towards the two hydrogel compartments hosting tumor cells that have undergone different treatments can be monitored and quantified. The particular competitive layout can be applied to investigate a plethora of different cancer biology phenotypes (e.g., drug-resistant vs aggressive, primary or metastatic, responders vs non-responders). Additionally, the gel embedded regions can be easily integrated with different cell populations to recreate more heterogenous TMEs, including stromal components (fibroblasts, endothelial cells)²³ or to simulate specific immunosuppressive milieu³⁴ (e.g., macrophages) for dissecting mechanisms of drug resistance and tumor evasion.
NOTE: Nuclear and active caspase staining, by using commercial kits for Live/dead assays (e.g., Thermo Fisher Scientific, Incucyte reagents), can be implemented to assess mitotic or apoptotic death events, as reported in Nguyen et al.³³.

1. Preparation of matrix solution with cells and Loading in the device
   NOTE: In the following experimental setting, the two gel regions contain mixtures of human A375M melanoma cell lines, grown in matrix solution (e.g., Matrigel), exposed to therapeutic agents used as monotherapy or in combination. This setting allowed us to evaluate the efficacy of a combination of two drugs with respect to single ones in a competitive fashion and to quantify their ability to attract PBMCs.
   1. Defrost a stock of matrix solution (e.g., Matrigel) by placing on ice into a 4 °C refrigerator one day before the experiment.
      NOTE: Do not expose the product to multiple freeze-thaw cycles as it becomes "clumpy". Other synthetic or natural hydrogels protocols can be suitable to be used in this setting. Please refer to ^33,34,35 for the preparation of cancer cells in collagen matrices.
   2. Resuspend A375 human melanoma cells, stained with live-compatible PKH67 Green Fluorescent Cell Linker in matrix solution (2 mg mL^-1). Where indicated, add 5-aza-2'-deoxycytidine (DAC; 2.5 µM), referred as DAC, and/or IFN-α2b ,referred as IFN, at the proper doses³².
      NOTE: Match the Lot # on the matrix bottle spec sheet. Based on the concentration calculate the volume of medium needed to make up to 2 mg mL^-1 Please adjust the optimal protein concentration and cancer cell suspension concentration accordingly to your application of interest.
   3. Pipette up and down carefully to avoid the generation of bubbles. Keep the microcentrifuge tube on ice while mixing to prevent any unwanted polymerization.
   4. After sterilization, place the devices on ice (using an ice bucket and lid) to avoid matrix solution solidification during the whole procedure of cell loading.
   5. Slowly inject the two IFN and DAC/IFN Matrigel/tumor cell mixtures (2-4 µL) into the left and right gel port, respectively with 10-µL micropipette using cold tips (Figure 5A). Apply gentle pressure to push matrix solution from one side until reaches the opposite one.
      NOTE: The volume of matrix solution was chosen to avoid overflowing into adjacent channels. Do not exert excessive pipetting pressure to prevent the solution from leaking into the media and central channels. If during loading gel path is blocked along the channel, try to insert the solution from the other inlet until the gel fronts meet. When removing the micropipette from the inlets, hold the plunger, otherwise the negative pressure will aspirate matrix solution.
   6. Place the device in an incubator in upright position at 37 °C and 5% CO₂ for 30 minutes to allow gelation of the matrix solution to take place (Figure 5B). Handle with care chips with embedded unpolymerized gel to prevent leaking out of the gel channel.
   7. In the meantime, resuspend PKH67-labelled PBMCs (1×10⁶ cells) in 10 µL of complete DMEM (Dulbecco's modified Eagle's medium).
   8. After matrix gelation, fill media channels with (50-100 µL) the same aliquot of culture medium in all six reservoirs to prevent gel drying in the chips. Keep in incubator until immune cell suspension seeding.
      NOTE: Check under a microscope the correct and homogeneous distribution of tumor cells in the gel and the integrity of the polymerized gel barriers. Partially or not uniform gelled regions or bubbles in the mixture lead to the premature flowing of PBMCs in gel media channels at the starting point of the experiment due to pressure initial fluctuations.
   9. Aspirate media from the six wells and position the tip near the inlet of a media channel to inject gently with moderate pressure PBMC cell suspension. The loading temporal sequence is depicted in Figure 5C:
      1. PBMCs in 10 µL medium into the upper central well.
      2. 50-100 µL medium into each of four wells of lateral channels.
      3. 40-90 µL medium into the upper central well.
      4. 50-100 µL medium into the lower central well.
   10. Ensure under a microscope that the PBMCs distribution remains confined in the central chamber after the loading step. (Figure 7A).
       NOTE: If it is not optimal, adjust the concentration if needed and repeat the seeding steps, using pristine chips. When calculating volumes for the planned experimental conditions, refer to an excess number of chips (15-20%) to take in account of potential errors and adjustments. Volumes and concentrations should be optimized according to the specific application.
   11. Place assembled devices on a level surface in the incubator at 37 °C and 5% CO₂ for subsequent fluorescence imaging acquisition. Handle with care chips after loading immune cells which are floating.
       NOTE: Compensate evaporative losses of volumes in reservoirs by replacing media every 2-3 days. For chemokine profiling, up to 100 µL from each of two wells of culture compartments can be aspirated, please refer to step 2.7, in step 1.
2. Automated Counting of recruited PBMCs in single-channel fluorescent images in ImageJ
   NOTE: Classical methods of immunofluorescence for confocal high-resolution imaging can be applied to on-chip operations as endpoint measurements. The basic staining procedure involves cell on-chip fixation, permeabilization, blocking, antibody binding, staining of nuclei with washing steps in between. Unlabeled immune cells infiltrated in 3D gels regions with embedded cancer microenvironments can be fixed at desired time-points and stained for expression markers of activation/exhaustion /maturation (e.g., for CD8 cells, monitoring of CD69, CD95, PD1, TIM3 markers). In Parlato et al.³⁵, phagocytosis of SW620 apoptotic cells was evaluated by confocal microscopy, using devices mounted on 170 µm-thick coverslips. IFN-DCs were stained adding on-chip anti-human HLA-DR-FITC Ab aliquots.
   To calculate the extent of infiltrated fluorescently stained live immune cells challenged with competitive signals, a common image analysis workflow is set as follows (Figure 7D-G):
   1. Acquire, at specific time endpoints, phase contrast, and red/green channels fluorescence microphotographs of the left and right gel regions containing tumor cells respectively exposed to single or combinations of pharmacological regimes.
      NOTE: Here images were captured by an EVOS-FL fluorescence microscope after cell loading (0 h), after 48 h and after 72 h of incubation (Figure 7A-B). A 4x-10x magnification was used to acquire the central chamber, the microchannel arrays, and the two juxtaposed side channels containing A375 plus IFN and A375 plus DAC/IFN.
      1. When performing acquisition operations, consider the parameters to measure and avoid saturation of features to be counted. Optimal segmentation results depend on the nature of the acquired images, due to variability in the biological samples themselves, quality of staining, and the microscopy techniques utilized for user-oriented applications.
   2. Load fluorescence single-channel data (in this case red = PBMCs), in Fiji by dragging it into the main window (Figure 7D). Duplicate the image to avoid overwriting the raw data during the selection of pre-processing filters until final segmentation.
      1. If the image is a color image (RGB), hit Image > Type 8 or 16-bit to convert to greyscale. Check that Edit > Options > Conversions is set to scale when converting.
   3. Preprocessing raw data via cleaning-up of noise and artifacts.
      1. Go to Process > Subtract Background menu by applying the rolling ball algorithm to correct uneven noise background with large spatial variations of intensities. Set the radius to at least the size of the largest foreground particle. Pick the Önizleme box for trial and error procedure to yield optimal results. Too small values may incorrectly remove structures of interest.
      2. In Brightness&Contrast command drag Minimum/Maximum sliders to change the range of intensities in the histogram. Shift the Maximum slider to the left to increase the brightness, without wash-out features. Move the Minimum slide to the right to increase the contrast of the image avoiding disappearance of less visible features in the background. Click Apply to fix changes.
   4. Image Enhancement.
      1. Go to Process > Filters and experiment with the Median, Gaussian filters on images (Figure 7E).
         NOTE: Pre-filtering radius should be adapted to the image noise pixels. The non-linear Median filter replaces pixel value with the median value of neighbours, to reduce salt-and-pepper noise. "Gaussian Blur" is used to smooth a digital picture, by replacing pixels with a weighted average of surrounding pixels. The weights come from the Gaussian probability distribution, so the nearest pixels are more influential.
      2. Go optionally to Process > Math > Gamma with ticked Önizleme box to increase the contrast.
         NOTE: Intensities set in the B&C panel are scaled between the two min and max limits. Values < 1.0 accentuate differences between low intensities while values > 1.0 accentuate differences between high intensities. Gamma correction is functional to find a display range, showing the dimmest objects without saturating the brightest.
   5. Creation of a binary image mask.
      1. Go to Image > Adjust > Threshold. The most simply employed method to determine thresholds relies on histogram analysis of intensity levels, as shown in Figure 7F. In the drop-menu, play with different global thresholding methods (in our case, Otsu is applied).
      2. Manually scroll or type a known range of pixel intensities in the histogram panels, observe the change of the red pattern overlaying the image which mostly resembles the actual cell area. The Reset button removes the overlay. Once satisfied, click Apply to generate a binary version of the image. Check Process > Binary > Options to control how thresholded images are displayed and how objects are identified by the Particle Analyzer.
   6. Use the menu command Process/Binary/Watershed Particles to divide partially overlapping or merged during the threshold. Watershed can often accurately cut them apart by adding a 1-pixel thick line. Perform morphological operations such as Dilate or Erode operations to either grow or remove pixels from under or over- saturated pixels.
      NOTE: For more information see the Menu Commands section or refer to MorphoLibJ, an integrated library based on mathematical morphology to process binary data.
   7. Quantitative Image Feature Description.
      1. Once obtained satisfactory object recognition, open the Particle Analyzer from Analyze > Analyze Particles. Particles can be excluded by their size and circularity, expressed in pixels or in a calibrated unit of measurement (check the correct scale relative to microscope settings under Image > Properties). To include everything, keep the default of 0-Infinity and circularity default range at 0.00 – 1.00 (0 = straight line, 1 = perfect circle).
      2. To filter small "noise" pixels or features of not interest, set the minimum and maximum range. Tick Include Holes, Show, Outline and Display Results options in the window field. Exclude on Edges will discard particles detected on the borders of the image. Add to Manager adds the obtained selection to the ROI manager for further analysis keeping the position information of the particle.
         ​NOTE: In the "ROI Manager", it is possible to correct automatic segmentation recorded output (merge split cells, split merged cells). Select, by using selection tools in the Fiji Toolbar, ROIs inside both gel regions where cancer cells are embedded to estimate immune infiltration.
   8. Export the obtained values to a spreadsheet to perform statistical analysis as shown in Figure 7G. "Results" lists a data table relative to numbered outlined particle properties identified. "Summarize" opens a window with the name of the image, total counts, and other information for the whole image.
      1. Go to Analyze > Set Measurements to include a wide range of parameters.
   9. Record optionally a macro (by selecting Plugins > Macros > Record) to automate the processing workflow and save time analysis on large datasets.
      1. Use the same processing routine to analyze green fluorescence channel in the same regions for analyzing morphological changes of tumor cells in 3D regions¹⁶