This research was supported by grants from the New Jersey Commission on Cancer Research (Pre-Doctoral Fellowship DFHS13PPCO17), the National Institutes of Health (CA049062), and the National Aeronautics and Space Administration (NNX15AD62G).

1. Preparation of Culture Media and Cells
<ol>
	<li>Prepare 500 ml of Eagle&#39;s minimum essential medium supplemented with 12.5% (vol/vol) heat-inactivated fetal bovine serum (FBS), 2 mM L-alanyl-L-glutamine, and 100 units of penicillin and 100 &#181;g of streptomycin per ml. 
	NOTE: The growth medium and supplement(s) can be easily exchanged for the growth requirements of other cell strains or cell lines.</li>
	<li>Prepare 70 &#181;l of cell culture medium for each insert (6-well format insert): Eagle&#39;s minimum essential medium supplemented with 50% (vol/vol) heat-inactivated FBS, 2 mM L-alanyl-L-glutamine, and 100 units of penicillin and 100 &#181;g of streptomycin per ml. 
	NOTE: The medium is supplemented with 50% FBS to facilitate cell attachment to the bottom side (i.e., underside) of the insert. The growth medium and supplement(s) can be easily exchanged for the growth requirements of other cell strains or cell lines.</li>
	<li>Prepare a cell suspension of the cells destined to be plated on the bottom side of the insert. 
	NOTE: Here, the results were obtained using AG1522 normal human fibroblasts grown in 75 cm2 cell culture flasks, and these cells will therefore be the focus of description of the cell suspension&#39;s preparation.</li>
	<li>To collect cells, remove the growth medium and wash the cell monolayer 2x with 5 ml 1x Phosphate Buffered Saline (PBS).</li>
	<li>Remove the PBS and spread 1 ml of 0.25% (vol/vol) trypsin with 2.21 mM ethylenediaminetetraacetic acid (EDTA), pre-warmed to room temperature (RT) over the cells for 2 min at RT (room temperature).</li>
	<li>Quench the trypsin activity with 9 ml of complete growth medium (prepared in step 1.1) and collect cells by gently pipetting the cell suspension over the surface of the flask 10x.</li>
	<li>Determine the cell concentration by using a hemocytometer or electronic counter and pipette the cell suspension volume that will provide 250,000 cells per insert into a sterile 15 ml centrifuge tube.</li>
	<li>Pellet the cell suspension by centrifugation (800 x g, 2 min). Suspend the cell pellet with growth medium (prepared in step 1.2) to create a concentration of 250,000 cells per 70 &#956;l. 
	NOTE: As cell culture in permeable microporous membrane inserts is based on 2D substrates, cell seeding is performed similarly, except the cells should be initially suspended in medium containing 50% (vol/vol) FBS to facilitate attachment.</li>
</ol>
2. Preparation of Inserts
NOTE: To ensure sterile conditions, work in a laminar flow biological safety cabinet dedicated to cell culture.
<ol>
	<li>Select inserts with a membrane pore size of 0.4 &#956;m, 1 &#956;m, or 3 &#956;m (determined by the experimental interest(s), as the pore size can be used to explore the role of different modes of intercellular communication). 
	NOTE: The 0.4 &#956;m pore is small enough to limit formation of functional gap junctions between cells on either side of the insert membrane and can be used to study intercellular communication by secreted factors. Whereas, the 1 &#956;m and 3 &#956;m pores are large enough to allow formation of functional gap junctions and can be used to study intercellular communication by both gap junctions and secreted factors.</li>
	<li>Remove individual inserts from the packaging and place into an equally sized multi-well dish. 
	NOTE: Inserts are available with various membrane surface areas to fit specific application needs (e.g., 6-well, 12-well, and 24-well inserts). Here, the results were obtained using the 6-well insert, and will therefore be the focus of the model description.</li>
	<li>Cover the dish, containing the inserts with the dish lid. Holding the multi-well dish with both hands, gently invert the dish such that the insert bottoms (i.e., underside) are now facing upwards.</li>
	<li>Remove the bottom of the multi-well dish, exposing the bottom side of the inserts. Working with one insert at a time, use a sterile pair of forceps and gently hold the insert in place. With the free hand, use a pipette with a wide-mouth tip to draw 70 &#956;l of the cell suspension (prepared in steps 1.3-1.8), containing 250,000 cells.</li>
	<li>To plate the cells, place the pipette tip gently on the center of the insert&#39;s membrane, and slowly pipette the 70 &#956;l of cell suspension while slowly moving the pipette tip around the surface of the membrane. Be careful not to extend the pipette tip and cell suspension to the edge of the insert. 
	NOTE: Touching the edge of the insert could result in loss of capillary tension of the cell suspension leading to the cells drying out during attachment.</li>
	<li>Repeat for remaining inserts, with care being exercised not to work directly over the exposed inserts to avoid potential microbial contamination.</li>
	<li>Gently return the multi-well dish bottom to cover the inserts. Keeping the dish with inserts inverted, incubate in a humidified air atmosphere of 5% (vol/vol) CO2 at 37 &#176;C for 30-45 min. 
	NOTE: If the cell suspension on the insert contacts the bottom of the well, carefully lift the dish bottom and rest it on the edge of the dish top, so that it forms a slight angle and creates greater separation between the surface of the dish and the bottom side of the inserts (where the cells are seeded). This will prevent the cells from attaching to the surface of the wells instead of the insert.</li>
	<li>Following the 30-45 min incubation time, gently remove the dish containing the inserts and place it in the laminar flow biological safety cabinet.</li>
	<li>With two hands and keeping the dish lid tight, gently re-invert the dish such that the bottom side of the inserts containing the attached cells now faces down.</li>
	<li>Slowly and carefully add 2 ml (1 ml for 3 &#956;m pore inserts to avoid migration of cells through the insert pores) of pre-warmed complete medium (see step 1.1) to each well, so that the bottom of the insert is immersed.</li>
	<li>After adding growth medium in all the wells containing inserts, place the dish back into the humidified incubator.</li>
	<li>Allow the inserts to remain undisturbed in the incubator for 48 hr. After 48 hr, feed the cells by aspirating the 2 ml of medium from the bottom of the well and adding 2 ml of fresh growth medium.</li>
	<li>After adding fresh medium to the bottom of the well, seed 1 ml of the second cell population suspension (~250,000 cells/ml) on the top side of the insert, which already has the first cell population growing on the bottom side (for these experiments, AG1522 (control) cells or cancer (experimental) cells are plated on the top of the inserts, which already have AG1522 cells, destined to become CAFs, growing on the bottom side of the inserts). Place the dish back into the humidified incubator. 
	NOTE: If working with cells that do not adhere well to the bottom of the insert, these cells can be alternatively grown on the top side of the insert.</li>
	<li>Change the medium at 24, 72, and 96 hr after seeding the second population of cells on the top side of the insert by aspirating the medium from the top of the insert and bottom of the well. Gently add 1 ml of fresh medium to the top side of the insert and 2 ml to the bottom of the well. Allow the two cell populations to remain in co-culture on either side of the permeable microporous insert membrane for 120 hr.</li>
</ol>
3. Collecting Cells from Insert by Trypsinization
NOTE: In addition to the ease of obtaining enriched cell populations, the insert TME model also allows for similar experimental treatments (e.g., incubation with chemicals, exposure to oxygen conditions that are above or below ambient atmosphere, ionizing radiation treatment, etc.) as other in vitro TME co-culture models. Furthermore, the permeable insert co-culture substrate can be analyzed by procedures already developed for standard 2D tissue culture models. For example, cells can be harvested from either side of the insert membrane to obtain highly enriched populations, which can then be utilized for analysis of endpoints (e.g., in situ immuno-detection, Western blotting) or propagated for subsequent experiments36.
<ol>
	<li>To collect cells, remove the insert from the growth medium and place it into a 35 mm cell culture dish containing 1 ml PBS. Wash the bottom and top sides of the insert 1x with 1 ml PBS.</li>
	<li>Remove the PBS and add 200 &#956;l of 0.25% (vol/vol) trypsin with 2.21 mM EDTA, pre-warmed to RT.
	<ol>
		<li>If collecting the cells grown on the bottom side of the insert, place the trypsin solution into the 35 mm dish and gently place the bottom of the insert into the trypsin solution for 2 min at RT.</li>
		<li>If collecting the cells grown on the top side of the insert, place the insert into a 35 mm dish and add the trypsin solution to the top of the insert and incubate for 2 min at RT.</li>
	</ol>
	</li>
	<li>Quench the trypsin activity by adding 800 &#956;l of complete growth medium.
	<ol>
		<li>If collecting the cells grown on the bottom of the insert, add the growth medium to the 35 mm dish and collect the cells by gently pipetting the 1 ml of cell suspension over the surface of the insert 10x, with the insert being held at a slight angle, such that the cell suspension collects into the dish.</li>
		<li>If collecting the cells from the top side of the insert, add the growth medium to the top side and gently pipette the 1 ml of cell suspension over the surface of the insert 10x.</li>
	</ol>
	</li>
</ol>
4. Characterization of Cell Purity by Flow Cytometry
NOTE: To characterize the ability of the permeable microporous membrane inserts to maintain the purity of the two cell populations (i.e., top and bottom) for up to 120 hr, sections 1-3 were performed, with green fluorescent protein (GFP)-tagged MDA-MB-231 cells being plated on the top side of the insert, and non-GFP-tagged AG1522 cells being plated on the bottom side of 0.4 &#181;m-, 1 &#181;m-, or 3 &#181;m pore inserts.
<ol>
	<li>Once the cells from the bottom side of the insert were collected (procedure described in Section 3), pellet the cell suspension by centrifugation (500 g, 30 sec).</li>
	<li>Remove the supernatant and suspend the cell pellet in 400 &#181;l of Hanks&#39; Balanced Salt Solution (HBSS) supplemented with 1% (vol/vol) FBS.</li>
	<li>Perform cytometric analysis on a flow cytometer equipped with a 488 nm laser to excite GFP and a bandpass filter of 530/30 to detect GFP photon emission37.</li>
	<li>For cell identification purposes, use AG1522 control cell populations to adjust forward and side scatter voltages on the flow cytometer. Adjust the GFP channel voltage to set the cellular autofluorescence value as the lower detection threshold for the GFP signal.</li>
	<li>Determine gating threshold using control cells. Assess purity of each cell population based upon the presence of GFP-positive cells versus control. 
	NOTE: A pure population would be reflective of no GPF-positive cells detected.</li>
</ol>
5. Characterization of Cells by In Situ Immunofluorescence
<ol>
	<li>Following trypsinization of cells from the insert (see section 3), collect cells, plate 5 x 104 cells in 250 &#956;l growth medium (see step 1.1) onto sterile glass coverslips, and allow to attach for 1 hr in a humidified air atmosphere of 5% (vol/vol) CO2 at 37 &#176;C incubator.</li>
	<li>At the end of the incubation, gently remove the dish containing the coverslip and place it in the laminar flow biological safety cabinet.</li>
	<li>Carefully add 2 ml of pre-warmed complete medium (see step 1.1) to each dish, so that the coverslip is immersed.</li>
	<li>After adding growth medium to all the dishes containing coverslips, return the dishes to a humidified air atmosphere of 5% (vol/vol) CO2 at 37 &#176;C in an incubator for 48 hr.</li>
	<li>Following the 48 hr incubation, wash the sample 3x with PBS and fix with 4% (wt/vol) formaldehyde in PBS for 10 min at RT.</li>
	<li>Rinse 5x with Tris Buffered Saline (TBS: 50 mM Tris-Cl, pH 7.5, 150 mM NaCl) and then permeabilize with 0.25% (vol/vol) Triton X-100, 0.1% (wt/vol) saponin for 5 min at RT.</li>
	<li>Block non-specific binding sites with 2% (vol/vol) normal goat serum (NGS), 1% (wt/vol) bovine serum albumin (BSA), 0.1% (vol/vol) Triton X-100 in TBS (blocking solution) for 1 hr at RT.</li>
	<li>Incubate with primary antibody (anti-Caveolin 1 (CAV1), 1:1,000) in blocking solution at 4 &#176;C overnight.</li>
	<li>Wash off unbound primary antibodies (3x, 5 min/wash) with 0.2% NGS (vol/vol), 1% (wt/vol) BSA, 0.1% (vol/vol) Triton X-100 in TBS (wash solution).</li>
	<li>Incubate with secondary antibody at RT for 1 hr in blocking solution (see section 5.4).</li>
	<li>Wash off unbound secondary antibody (3x, 5 min/wash) with wash solution (see step 5.6).</li>
	<li>Mount coverslips on a slide with an anti-fade mounting medium containing 4&#39;,6-diamidino-2-phenylindole (DAPI), and seal with nail polish.</li>
	<li>Observe cells using a 63X oil magnification objective on an inverted microscope equipped with an external light source for fluorescence excitation, and capture images using an attached digital camera for subsequent analysis.</li>
</ol>
6. Characterization of Cells by Western Blot
NOTE: The Western blot procedure is described elsewhere38. A brief outline is described here:
<ol>
	<li>Following detachment of cells from the insert by trypsinization (see section 3), pellet the cell suspension by centrifugation (500 g, 30 sec).</li>
	<li>Remove supernatant and suspend the cell pellet 2x with 100 &#181;l PBS.</li>
	<li>Remove supernatant and lyse cells in 50 &#181;l of modified radioimmunoprecipitation assay (RIPA) buffer (Tris pH 7.5 50 mM, NaCl 150 mM, NP40 1% (vol/vol), sodium deoxycholate monohydrate (DOC) 0.5% (vol/vol), sodium dodecyl sulfate (SDS) 0.1% (vol/vol)), containing protease and phosphatase inhibitor cocktail.</li>
	<li>Incubate for 20 min on ice and centrifuge at 19,000 g for 15 min at 4 &#176;C.</li>
	<li>Determine concentration of proteins in the supernatant using a protein optical density assay, compatible with the detergents in the RIPA buffer.</li>
	<li>Separate proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroblot onto a 0.2 &#181;m nitrocellulose membrane.</li>
	<li>Incubate membranes in TBS containing 0.1% (vol/vol) Tween-20 (TBST) and 4% (wt/vol) skim milk (blocking solution) for 60 min and react with primary antibody at 4 &#176;C overnight (anti-CAV1, 1:1,000).</li>
	<li>Wash membrane 3x with TBST at RT (5 min/wash).</li>
	<li>Incubate membranes for 30 min in blocking solution containing an anti-mouse secondary antibody conjugated with horseradish peroxidase (HRP) (1:5,000).</li>
	<li>Wash membrane 3x with TBST at RT (5 min/wash).</li>
	<li>Visualize reaction products by chemiluminescence using a Western blotting detection system.</li>
</ol>
7. Characterization of Intercellular Communication by Flow Cytometry
NOTE: To characterize the adaptability of the permeable microporous membrane inserts to examine different modes of intercellular communication (e.g., secreted factors, gap junctions). Sections 1-2.12 were performed with non-fluorescent AG1522 cells being plated on the bottom side of 0.4 &#181;m-, 1 &#181;m-, or 3 &#181;m-pore inserts.
<ol>
	<li>Culture non-labeled AG1522 cells in a 35 mm dish to 90% confluency using medium described in section 1.1.</li>
	<li>Rinse cells 1x with 1 ml of HBSS.</li>
	<li>Load cells with Calcein AM by incubating cells in HBSS containing 30 &#181;M of Calcein AM.</li>
	<li>Incubate for 15 min in a humidified air atmosphere of 5% (vol/vol) CO2 at 37 &#176;C.</li>
	<li>Wash cells 2x with 1 ml of HBSS.</li>
	<li>Trypsinize cells by adding 200 &#181;l solution of 0.25% (vol/vol) trypsin with 2.21 mM EDTA for 2 min at RT.</li>
	<li>Quench the trypsin activity by adding 800 &#181;l of complete medium containing 12.5% (vol/vol) FBS.</li>
	<li>Bring cells into suspension by repeated pipetting.</li>
	<li>Determine cell concentration by using a hemocytometer or electronic counter and plate 250,000 cells loaded with Calcein AM to the top side of inserts that already have AG1522 cells growing on the bottom side.</li>
	<li>Incubate the inserts in a humidified air atmosphere of 5% CO2 at 37 &#176;C for 6 hr.</li>
	<li>After 6 hr, collect cells from the bottom side of the inserts, as described in section 3.</li>
	<li>Pellet the cell suspension by centrifugation (500 g, 30 sec).</li>
	<li>Remove the supernatant and suspend the cell pellet in 400 &#181;l of HBSS supplemented with 1% (vol/vol) FBS.</li>
	<li>Analyze the cells on a cytometer equipped with a laser and filter capable of exciting and detecting the emission of Calcein (496 nm and 516 nm, respectively), per manufacturer protocol.</li>
	<li>For cell identification purposes, use an unstained AG1522 control cell population to adjust forward and side scatter voltages on the flow cytometer. Adjust the Calcein channel voltage to set the cellular autofluorescence value as the lower detection threshold for the Calcein signal.</li>
	<li>Determine the upper threshold limit using Calcein-loaded control cells. Assess communication for each cell insert based upon the presence of Calcein-positive cells versus control.</li>
</ol>

The authors declare that they have no competing or conflicting interests.

The protocol described here is a simple, adaptable in vitro procedure (Figure 1) that utilizes a permeable microporous membrane insert to generate highly enriched individual cell populations from a co-culture of heterotypic cells. Significantly, the model is suitable for investigating various modes of intercellular communication. The critical steps include selecting the appropriate pore-size insert for specific experimental interest(s), seeding the first cell population on the bottom side of the...

The tumor microenvironment (TME) is a highly complex system comprised of carcinoma cells that co-exist and evolve alongside host stroma. This stromal component typically consists of fibroblasts, myofibroblasts, endothelial cells, various immune components, as well as an extracellular matrix1. A significant constituent, often the majority of this stroma, are activated fibroblasts, frequently referred to as cancer-associated fibroblasts or carcinoma-associated fibroblasts (CAF)2,3. Unlike normal, non-activated fibroblasts, CAFs contribute to tumor initiation, progression, angiogenesis, invasion, metastasis, and recurrence4-11 in a wide variety of carcinomas, including breast, prostate, lung, pancreas, skin, colon, esophagus, and ovary5,6,12-17. Yet, the exact nature of the contribution of CAFs throughout cancer pathogenesis remains poorly defined. Furthermore, clinical evidence has demonstrated a prognostic value of CAFs, correlating their presence to high-grade malignancies, therapy failure, and overall poor prognosis10,18,19.
Clearly, enhancing our understanding of the initiating events in CAF development, as well as the intercellular communications mediating their role within the TME, may provide exciting new therapeutic targets and enhanced strategies that could improve patient outcomes. Towards this goal, several in vivo and in vitro models have been developed. While in vivo approaches are more reflective of patients&#39; TME, they possess limitations, including the immense complexity and heterogeneity both within and between tumors. Furthermore, tumor samples from human subjects often represent highly developed TME and do not permit an understanding of the TME initiating events. Experimental animal studies offer some advantages, however generalization of animal data to humans should be done with caution due to differences in physiology between humans and animals such as rodents (e.g., thiol chemistry20, metabolic rate21, tolerance to stress22, etc.). Further, unlike the human population, which is genetically heterogeneous in nature, laboratory animals are typically bred to homogeneity. Also, it is often difficult to examine transient physiological variations and cell phenotype changes, as well as to control for specific experimental parameters using animals such as rodents. Thus, in vitro 2- and 3-dimensional (2D and 3D) tissue culture models are frequently utilized to advance the basic understanding of TME development. In spite of their lack of an accurate portrayal of the complexity of in vivo systems, these models offer advantages that greatly facilitate mechanistic investigations. In vitro models allow for a more simplified, focused, and cost-effective analysis of the TME, whereby statistically significant data can be generated in cells free of systemic variations that arise in animals.
There are several varieties of in vitro systems. The two most commonly used TME in vitro models consist of mixed monolayer or spheroid cell cultures. Both culture methods are advantageous for basic studies of intercellular interactions (e.g., normal cells with tumor cells) and for the analysis of various TME specific cell phenotype changes (e.g., emergence of cancer-associated fibroblasts from normal fibroblasts). Additionally, the spheroids are able to create a more reflective tissue-like structure of the TME, and can be representative of tumor heterogeneity23. However spheroids often produce widely varying oxygen tension gradients across layers, which may complicate experimental conclusions24. Unfortunately, both models are extremely limited in their ability to isolate pure cell populations for further characterization and study following co-culture. To do so would require at least one cell type to be fluorescently-tagged or labeled with an identifying maker, and then subjecting the mixed co-culture to extensive processing and cell sorting to separate the cell populations. While a cell sorter is capable of isolating a rather pure cell population, one must be cognizant of cellular stress and potential microbial contamination risks25.
To facilitate the understanding of intercellular communication, great efforts have been devoted towards developing and optimizing in vitro systems that closely mimic the in vivo environment, while permitting a simplified approach. One such tool is the permeable microporous insert, a membrane substrate that was first developed in 195326 and subsequently adapted for diverse applications and studies (e.g., cell polarity27, endocytosis28, drug transport29, tissue modeling30, fertilization31, bystander effect32,33, etc.). This system permits the growth of cells with in vivo-like anatomical and functional differentiation, as well as expression of many in vivo markers34,35 that are not observed when cultured on impermeable plasticware. Furthermore, the extremely thin porous membrane (10 &#181;m thick) permits rapid diffusion of molecules and equilibration times, which simulates the in vivo environment and permits independent cellular functioning at both the apical and basolateral cell domains. An additional advantage of the insert&#39;s utility as a TME system is its physical separation of two heterotypic cell populations grown on either side of the membrane in the same environmental conditions, while maintaining various modes of intercellular communication through the membrane pores. Though physically separated, the two cell populations are metabolically coupled via secreted elements and, as described here, also through gap-junctional channels. Additionally, by maintaining the inserts at in vivo partial oxygen tension (PO2), the model reduces the complications of oxygen and chemical gradients observed in other systems. Rather, it increases the understanding of natural mechanisms controlling the TME. Notably, the two cell populations can be easily isolated with high purity, without fluorescent tagging and/or cell sorting following extended periods of co-culture.
Here we describe an in vitro TME protocol consisting of human breast carcinoma cells and human fibroblasts grown, respectively, on either side of a permeable microporous membrane insert, but yet in continuous bi-directional communication through the membrane pores. We show that by using membranes with different pore sizes, the contribution of a specific type of intercellular communication (e.g., secreted factors versus gap junctions) to the development of the TME can be investigated.

<table><tbody><tr><td>&lt;strong&gt;For Cell Culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>AG01522 (&lt;em&gt;i.e.&lt;/em&gt;, AG1522) human diploid fibroblast</td><td>Coriell</td><td>107661</td><td>Passage 8-13</td></tr><tr><td>MDA-MB-231-luc-D3H1 breast adenocarcinoma cell line</td><td>PerkinElmer</td><td>119261</td><td>Parental line: ATCC (#HTB-26)</td></tr><tr><td>MDA-MB-231/GFP breast adenocarcinoma cell line</td><td>Cell Biolabs</td><td>AKR-201</td><td></td></tr><tr><td>Eagle&#039;s minimal essential medium (MEM)</td><td>Corning Cellgro</td><td>15-010-CV</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS), Qualified</td><td>Sigma</td><td>F6178-500mL</td><td></td></tr><tr><td>Corning Glutagro Supplement (200 mM L-alanyl-L-glutamine)</td><td>Corning Cellgro</td><td>25-015-Cl</td><td></td></tr><tr><td>Penicillin Streptomycin Solution, 100x</td><td>Corning Cellgro</td><td>30-002-Cl</td><td></td></tr><tr><td>Transwell Insert (&lt;em&gt;i.e.&lt;/em&gt;, permeable microporous membrane insert) (0.4 &amp;mu;m pore)</td><td>Costar</td><td>3450</td><td></td></tr><tr><td>Transwell Insert (&lt;em&gt;i.e.&lt;/em&gt;, permeable microporous membrane insert) (1 &amp;mu;m pore)</td><td>Greiner bio-one</td><td>657610</td><td></td></tr><tr><td>Transwell Insert (&lt;em&gt;i.e.&lt;/em&gt;, permeable microporous membrane insert) (3 &amp;mu;m pore)</td><td>Costar</td><td>3452</td><td></td></tr><tr><td>6-well Culture Plate</td><td>Greiner Bio-One Cellstar</td><td>657160-01</td><td></td></tr><tr><td>75 cm&lt;sup&gt;2&lt;/sup&gt; cell culture flask</td><td>CellStar</td><td>658 170</td><td></td></tr><tr><td>Phosphate-Buffered Saline (PBS), 1x</td><td>Corning Cellgro</td><td>21-040-CV</td><td>without calcium &amp;amp; magnesium</td></tr><tr><td>0.25% (vol/vol) Trypsin, 2.21 mM EDTA, 1x</td><td>Corning Cellgro</td><td>25-053-Cl</td><td></td></tr><tr><td>15 ml Centrifuge Tube</td><td>CellTreat</td><td>229411</td><td></td></tr><tr><td>35 mm x 10 mm Cell Culture Dish</td><td>Greiner bio-one</td><td>627 160</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;For Immunofluorescent Microscopy&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Mouse anti-Caveolin 1</td><td>BD Transduction Laboratories</td><td>610406</td><td>&lt;em&gt;In situ&lt;/em&gt; Immunofluorescence - 1:5,000</td></tr><tr><td>Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate</td><td>ThermoFisher Scientific</td><td>A-11029</td><td>&lt;em&gt;In situ&lt;/em&gt; Immunofluorescence - 1:2,000</td></tr><tr><td>Bovine Serum Albumin - Fraction V</td><td>Rockland</td><td>BSA-50</td><td>Immunoglobulin and protease free</td></tr><tr><td>16% (wt/vol) Formaldehyde Solution</td><td>ThermoFisher Scientific</td><td>28908</td><td>Dilute to 4% with 1x PBS</td></tr><tr><td>Premium Cover Glass (22 mm x 22 mm No.1)</td><td>Fisher</td><td>12548B</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787-50ML</td><td></td></tr><tr><td>SlowFade Gold antifade reagent with DAPI</td><td>Invitrogen</td><td>S36938</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;For Flow Cytometric Analysis&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Calcein, AM</td><td>Molecular Probes</td><td>C3100MP</td><td></td></tr><tr><td>Hanks&#039; Balanced Salt Solution (HBSS)</td><td>Gibco</td><td>14025-076</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;For Western Blot Analysis&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Mouse anti-Caveolin 1</td><td>BD Transduction Laboratories</td><td>610406</td><td>Western Blot - 1:1,000</td></tr><tr><td>Tween-20</td><td>BioRad</td><td>170-6531</td><td></td></tr><tr><td>Nitrocellulose Membrane (0.2 &amp;mu;m)</td><td>BioRad</td><td>162-0112</td><td></td></tr><tr><td>Western Lightning Plus-ECL</td><td>PerkinElmer</td><td>NEL104001EA</td><td></td></tr><tr><td>BioRad DC Protein Assay</td><td>BioRad</td><td>500-0116</td><td></td></tr><tr><td>Sodium dodecyl sulfate (SDS)</td><td>BioRad</td><td>161-0302</td><td></td></tr><tr><td>Sodium deoxycholate monohydrate (DOC)</td><td>Sigma</td><td>D5670</td><td></td></tr><tr><td>IGEPAL CA-630 (NP40)</td><td>Sigma</td><td>I8896</td><td></td></tr><tr><td>30% Acrylamide/Bis Solution, 37.5:1</td><td>BioRad</td><td>161-0158</td><td></td></tr></tbody></table>

a mimic of the tumor microenvironment: a simple method for generating enriched cell populations and investigating intercellular communication

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events leading to stromal activation and establishment of the tumor microenvironment (TME). Several in vitro and in vivo models of the TME have been developed; however, in general these models do not readily permit isolation of individual cell populations, under non-perturbing conditions, for further study. To circumvent this difficulty, we have employed an in vitro TME model using a cell growth substrate consisting of a permeable microporous membrane insert that permits simple generation of highly enriched cell populations grown intimately, yet separately, on either side of the insert&#39;s membrane for extended co-culture times. Through use of this model, we are capable of generating greatly enriched cancer-associated fibroblast (CAF) populations from normal diploid human fibroblasts following co-culture (120 hr) with highly metastatic human breast carcinoma cells, without the use of fluorescent tagging and/or cell sorting. Additionally, by modulating the pore-size of the insert, we can control for the mode of intercellular communication (e.g., gap-junction communication, secreted factors) between the two heterotypic cell populations, which permits investigation of the mechanisms underlying the development of the TME, including the role of gap-junction permeability. This model serves as a valuable tool in enhancing our understanding of the initial events leading to cancer-stroma initiation, the early evolution of the TME, and the modulating effect of the stroma on the responses of cancer cells to therapeutic agents.

Here we adapted a permeable microporous membrane insert to develop an in vitro heterotypic cell co-culture system that mimics the in vivo tumor microenvironment (Figure 1). This system allows for two different cell populations to be grown on either side of the insert&#39;s porous-membrane for extended periods of time (up to 120 hr, in our use). Importantly the system is capable of maintaining the purity of the cell populations, as determined by plating G...

Watch this Scientific Journal Video about A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication at JoVE.com

A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

We adapted a permeable microporous membrane insert to mimic the tumor microenvironment (TME). The model consists of a mixed cell culture, allows simplified generation of highly enriched individual cell populations without using fluorescent tagging or cell sorting, and permits studying intercellular communication within the TME under normal or stress conditions.

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events ...

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10.2K Views.  Rutgers University. We adapted a permeable microporous membrane insert to mimic the tumor microenvironment (TME). The model consists of a mixed cell culture, allows simplified generation of highly enriched individual cell populations without using fluorescent tagging or cell sorting, and permits studying intercellular communication within the TME under normal or stress conditions.

Video: A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract (ECME)-based Three-dimensional System

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, thus greatly influencing normal tissue homeostasis and disease outcome. In breast cancer, tumor-associated macrophages (TAMs) play a critical role in disease progression, metastasis, and recurrence; therefore, understanding the mechanisms of monocyte chemoattraction to the tumor microenvironment and their interactions with tumor cells is important to control the disease. Here, we provide a detailed description of a three-dimensional (3D) co-culture system of human breast cancer (BrC) cells and human monocytes. BrC cells produced high basal levels of regulated on-activation, normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), and granulocyte-macrophage colony-stimulating factor (G-CSF), while in co-culture with monocytes, pro-inflammatory cytokines Interleukin (IL)-1 beta (IL-1&#946;) and IL-8 were enriched together with matrix metalloproteinases (MMP)-1, MMP-2, and MMP-10. This tumor stroma microenvironment promoted resistance to anoikis in MCF-10A 3D acini-like structures, chemoattraction of monocytes, and invasion of aggressive BrC cells. The protocols presented here provide an affordable alternative to study intra-tumor communication and are an example of the great potential that in vitro 3D cell systems provide to interrogate specific features of tumor biology related to tumor aggression.

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System

Here, we describe a three-dimensional culture method to analyze the morphology of primary breast cancer cells, as well as to study their direct/indirect interactions with monocytes and the outcomes such as collagen degradation, immune cell recruitment, cell invasion, and promotion of cancer-related inflammation.

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, ...

Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for immunotherapies. Recent clinical advances in immunotherapy highlight the need for reproducible methods to accurately and thoroughly characterize the tumor and its associated immune infiltrate. Tumor enzymatic digestion and flow cytometric analysis allow broad characterization of numerous immune cell subsets and phenotypes; however, depth of analysis is often limited by fluorophore restrictions on panel design and the need to acquire large tumor samples to observe rare immune populations of interest. Thus, we have developed an effective and high throughput method for separating and enriching the tumor immune infiltrate from the non-immune tumor components. The described tumor digestion and centrifugal density-based separation technique allows separate characterization of tumor and tumor immune infiltrate fractions and preserves cellular viability, and thus, provides a broad characterization of the tumor immunologic state. This method was used to characterize the extensive spatial immune heterogeneity in solid tumors, which further demonstrates the need for consistent whole tumor immunologic profiling techniques. Overall, this method provides an effective and adaptable technique for the immunologic characterization of subcutaneous solid murine tumors; as such, this tool can be used to better characterize the tumoral immunologic features and in the preclinical evaluation of novel immunotherapeutic strategies.

Here we describe a method to separate and enrich components of the tumor immune and non-immune microenvironment in established subcutaneous tumors. This technique allows for the separate analysis of tumor immune infiltrate and non-immune tumor fractions which can permit comprehensive characterization of the tumor immune microenvironment.

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for ...

Using Microarrays to Interrogate Microenvironmental Impact on Cellular Phenotypes in Cancer

Understanding the impact of the microenvironment on the phenotype of cells is a difficult problem due to the complex mixture of both soluble growth factors and matrix-associated proteins in the microenvironment in vivo. Furthermore, readily available reagents for the modeling of microenvironments in vitro typically utilize complex mixtures of proteins that are incompletely defined and suffer from batch to batch variability. The microenvironment microarray (MEMA) platform allows for the assessment of thousands of simple combinations of microenvironment proteins for their impact on cellular phenotypes in a single assay. The MEMAs are prepared in well plates, which allows the addition of individual ligands to separate wells containing arrayed extracellular matrix (ECM) proteins. The combination of the soluble ligand with each printed ECM forms a unique combination. A typical MEMA assay contains greater than 2,500 unique combinatorial microenvironments that cells are exposed to in a single assay. As a test case, the breast cancer cell line MCF7 was plated on the MEMA platform. Analysis of this assay identified factors that both enhance and inhibit the growth and proliferation of these cells. The MEMA platform is highly flexible and can be extended for use with other biological questions beyond cancer research.

The purpose of the method presented here is to show how microenvironment microarrays (MEMA) can be fabricated and used to interrogate the impact of thousands of simple combinatorial microenvironments on the phenotype of cultured cells.

Understanding the impact of the microenvironment on the phenotype of cells is a difficult problem due to the complex mixture of both soluble growth ...

Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions contribute to tissue architectural changes observed during tumorigenesis remains challenging due to the lack of appropriate models. In this protocol, we describe the generation of a 3D coculture model using a LUSC primary cell culture known as TUM622. TUM622 cells were established from a LUSC patient-derived xenograft (PDX) and have the unique property to form acinar-like structures when seeded in a basement membrane matrix. We demonstrate that TUM622 acini in 3D coculture recapitulate key features of tissue architecture during LUSC progression as well as the dynamic interactions between LUSC cells and components of the tumor microenvironment (TME), including the extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs). We further adapt our principal 3D culturing protocol to demonstrate how this system could be utilized for various downstream analyses. Overall, this organoid model creates a biologically rich and adaptable platform that enables one to gain insight into the cell-intrinsic and extrinsic mechanisms that promote the disruption of epithelial architectures during carcinoma progression and will aid the search for new therapeutic targets and diagnostic markers.

An in vitro model system was developed to capture tissue architectural changes during lung squamous carcinoma (LUSC) progression in a 3-dimensional (3D) co-culture with cancer-associated fibroblasts (CAFs). This organoid system provides a unique platform to investigate the roles of diverse tumor cell-intrinsic and extrinsic changes that modulate the tumor phenotype.

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions ...

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

Flow Cytometry-Based Monitoring of Cancer-Immunity Cycle 

Co-Culture In Vitro Systems to Reproduce the Cancer-Immunity Cycle

Fundamental cancer research and the development of effective counterattack therapies both rely on experimental studies detailing the interactions between cancer and immune cells, the so-called cancer-immunity cycle. In vitro co-culture systems combined with multiparametric flow cytometry (mFC) and tumor-on-a-chip microfluidic devices (ToCs) enable simple, fast, and reliable monitoring and characterization of each step of the cancer-immunity cycle and lead to the identification of the mechanisms responsible for tipping the balance between cancer immunosurveillance and immunoevasion. A thorough understanding of the dynamic interplays between cancer and immune cells provides critical insights to outsmart tumors and will accelerate the pace of therapeutic personalization and optimization in patients. Specifically, here we detail a straightforward mFC- and ToC-assisted protocol for unraveling the dynamic complexities of each step of the cancer-immunity cycle in murine cancer cell lines and mouse-derived immune cells and focus on immunosurveillance. Considering the time- and cost-related features of this protocol, it is certainly feasible on a large scale. Moreover, with minor variations, this protocol can be both adapted to human cancer cell lines and human peripheral-blood-derived immune cells and combined with genetic and/or pharmacologic inhibition of specific pathways in order to identify biomarkers of immune response.

Monitoring the Cancer-Immunity Cycle and Exploring Tumor Microenvironment Dynamics

Here, we detail a simple, fast, and reliable multiparametric flow cytometry- and tumor-on-a-chip-assisted protocol to monitor and characterize the steps constituting the cancer-immunity cycle. Indeed, a thorough understanding of the interplays between cancer and immune cells provides critical insights to outsmart tumors and guide clinical care.

Fundamental cancer research and the development of effective counterattack therapies both rely on experimental studies detailing the interactions between ...

Renal Cancer and Healthy Tissue Co-Culture Using Microphysiological Systems

Microfluidic Co-culture of Renal Healthy and Tumor Epithelium to Model Kidney Cancer Progression

Microphysiological systems (MPS) have enabled the introduction of more complex and relevant physiological elements into in vitro models, recreating intricate morphological features in three-dimensional environments with dynamic interactions lacking in conventional models. We implemented a renal cell carcinoma (RCC) co-culture model to recreate the cross-talk between healthy and malignant renal tissue. 
This model is based on the referenced multi-organ platform and consists of co-culturing a reconstructed renal proximal tubule with RCC spheroids. Custom-designed 3D-printed chambers were used to culture human renal epithelial proximal tubule cells (RPTEC) and facilitate their self-assembly into a renal tubule contained in a collagen type I matrix. Caki-1 RCC cells were embedded in an agar collagen matrix, subsequently forming cancer spheroids. Both collagen and agar/collagen gels were optimized to maintain their integrity during cyclic perfusion and withstand shear stress during a minimum culture period of 7 days. 
The gels also enable adequate nutrient supply and cell secretions. Moreover, the agar/collagen gels limit the overproliferation of RCC cells, ensuring relatively homogeneous spheroid size. The MPS chip microfluidic circuits comprise two independent culture chambers with the size of a standard 96-microplate well. The renal tubule and RCC gels populate separate chambers and share the same culture media, which is recirculated approximately twice per minute. Under these conditions, we observed upregulation of immune factor expression and secretion in the renal tubules (interleukin-8 and tumor necrosis factor-alpha). The renal tubules also shift their metabolic activity towards glycolysis under the influence of RCC. This novel approach demonstrates that a co-culture-based MPS can amplify the complexity of RCC in vitro and be employed to study the impact of cancer on non-tumor cells.

Here, we present protocols that detail instructions for implementing 3D cell cultures using collagen and collagen-agarose matrixes in a microphysiological system. These protocols support renal proximal tubule and renal cell carcinoma spheroids co-culture, simulating in vivo conditions and enabling advanced investigation of kidney cancer cell interactions.

Microphysiological systems (MPS) have enabled the introduction of more complex and relevant physiological elements into in vitro models, recreating ...

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids and patient-derived xenograft organoids are pathophysiologically relevant, they are costly to propagate, difficult to manipulate, and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. 
Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. The advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these heteromulticellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells (THP-1), and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. 
Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent with the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, heteromulticellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in heteromulticellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression.

There is a critical need for 3D cancer models that capture heterocellular crosstalk to study cancer metastasis. Our study presents the generation of heteromulticellular stromal-epithelial in a scaffold and scaffold-free environment that can be used to study invasion and cellular spatial distributions.

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Medicine

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

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise invisible processes. Advanced cell assays closely mimicking in vivo scenery are establishing themselves as novel ways to visualize and measure the bidirectional tumor-host interplay influencing the progression of cancer. Here we describe two versatile protocols to recreate highly controllable 2D and 3D co-cultures in microdevices, mimicking the complexity of the tumor microenvironment (TME), under natural and therapy-induced immunosurveillance. In section 1, an experimental setting is provided to monitor crosstalk between adherent tumor cells and floating immune populations, by bright field time-lapse microscopy. As an applicative scenario, we analyze the effects of anti-cancer treatments, such as the so-called immunogenic cancer cell death inducers on the recruitment and activation of immune cells. In section 2, 3D tumor-immune microenvironments are assembled in a competitive layout. Differential immune infiltration is monitored by fluorescence snapshots up to 72 h, to evaluate combination therapeutic strategies. In both settings, image processing steps are illustrated to extract a plethora of immune cell parameters (e.g., immune cell migration and interaction, response to therapeutic agents). These simple and powerful methods can be further tailored to simulate the complexity of the TME encompassing the heterogeneity and plasticity of cancer, stromal and immune cells subtypes, as well as their reciprocal interactions as drivers of cancer evolution. The compliance of these rapidly evolving technologies with live-cell high-content imaging can lead to the generation of large informative datasets, bringing forth new challenges. Indeed, the triangle &#39;&#39;co-cultures/microscopy/advanced data analysis&#34; sets the path towards a precise problem parametrization that may assist tailor-made therapeutic protocols. We expect that future integration of cancer-immune on-a-chip with artificial intelligence for high-throughput processing will synergize a large step forward in leveraging the capabilities as predictive and preclinical tools for precision and personalized oncology.

In the age of immunotherapy and single-cell genomic profiling, cancer biology requires novel in vitro and computational tools for investigating the tumor-immune interface in a proper spatiotemporal context. We describe protocols to exploit tumor-immune microfluidic co-cultures in 2D and 3D settings, compatible with dynamic, multiparametric monitoring of cellular functions.

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise ...