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 border="0" cellpadding="0" cellspacing="0" width="886">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">For Cell Culture</td>
			<td style="width:180px;"></td>
			<td style="width:139px;"></td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">AG01522 (i.e., AG1522) human diploid fibroblast</td>
			<td style="width:180px;">Coriell</td>
			<td style="width:139px;">107661</td>
			<td style="width:279px;">Passage 8-13</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">MDA-MB-231-luc-D3H1 breast adenocarcinoma cell line</td>
			<td style="width:180px;">PerkinElmer</td>
			<td style="width:139px;">119261</td>
			<td style="width:279px;">Parental line: ATCC (#HTB-26)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">MDA-MB-231/GFP breast adenocarcinoma cell line</td>
			<td style="width:180px;">Cell Biolabs</td>
			<td style="width:139px;">AKR-201</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Eagle&#39;s minimal essential medium (MEM)</td>
			<td style="width:180px;">Corning Cellgro</td>
			<td style="width:139px;">15-010-CV</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Fetal Bovine Serum (FBS), Qualified</td>
			<td style="width:180px;">Sigma</td>
			<td style="width:139px;">F6178-500mL</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">Corning Glutagro Supplement (200mM L-alanyl-L-glutamine)</td>
			<td style="width:180px;">Corning Cellgro</td>
			<td style="width:139px;">25-015-Cl</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Penicillin Streptomycin Solution, 100X</td>
			<td style="width:180px;">Corning Cellgro</td>
			<td style="width:139px;">30-002-Cl</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:289px;">Transwell Insert (i.e., permeable microporous membrane insert) (0.4 &#956;m pore)</td>
			<td style="width:180px;">Costar</td>
			<td style="width:139px;">3450</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:289px;">Transwell Insert (i.e., permeable microporous membrane insert) (1 &#956;m pore)</td>
			<td style="width:180px;">Greiner bio-one</td>
			<td style="width:139px;">657610</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:289px;">Transwell Insert (i.e., permeable microporous membrane insert) (3 &#956;m pore)</td>
			<td style="width:180px;">Costar</td>
			<td style="width:139px;">3452</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">6-well Culture Plate</td>
			<td style="width:180px;">Greiner Bio-One Cellstar</td>
			<td style="width:139px;">657160-01</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">75 cm2 cell culture flask</td>
			<td style="width:180px;">CellStar</td>
			<td style="width:139px;">658 170</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Phosphate-Buffered Saline (PBS), 1X</td>
			<td style="width:180px;">Corning Cellgro</td>
			<td style="width:139px;">21-040-CV</td>
			<td style="width:279px;">without calcium &#38; magnesium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">0.25% (vol/vol) Trypsin, 2.21 mM EDTA, 1X</td>
			<td style="width:180px;">Corning Cellgro</td>
			<td style="width:139px;">25-053-Cl</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">15 mL Centrifuge Tube</td>
			<td style="width:180px;">CellTreat</td>
			<td style="width:139px;">229411</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">35 x 10 mm Cell Culture Dish</td>
			<td style="width:180px;">Greiner bio-one</td>
			<td style="width:139px;">627 160</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Name</td>
			<td style="width:180px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">For Immunofluorescent Microscopy</td>
			<td style="width:180px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">Mouse anti-Caveolin 1</td>
			<td style="width:180px;">BD Transduction Laboratories</td>
			<td style="width:139px;">610406</td>
			<td style="width:279px;">In situ Immunofluorescence - 1:5000</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate</td>
			<td style="width:180px;">ThermoFisher Scientific</td>
			<td style="width:139px;">A-11029</td>
			<td>In situ Immunofluorescence - 1:2000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Bovine Serum Albumin - Fraction V</td>
			<td style="width:180px;">Rockland</td>
			<td style="width:139px;">BSA-50</td>
			<td>Immunoglobulin and protease free</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">16% (wt/vol) Formaldehyde Solution</td>
			<td style="width:180px;">ThermoFisher Scientific</td>
			<td style="width:139px;">28908</td>
			<td>Dilute to 4% with 1X PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Premium Cover Glass (22x22 mm No.1)</td>
			<td style="width:180px;">Fisher</td>
			<td style="width:139px;">12548B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Triton X-100</td>
			<td style="width:180px;">Sigma</td>
			<td style="width:139px;">T8787-50ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">SlowFade Gold antifade reagent with DAPI</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:139px;">S36938</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Name</td>
			<td style="width:180px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">For Flow Cytometric Analysis</td>
			<td style="width:180px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Calcein, AM</td>
			<td style="width:180px;">Molecular Probes</td>
			<td style="width:139px;">C3100MP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Hanks&#39; Balanced Salt Solution (HBSS)</td>
			<td style="width:180px;">Gibco</td>
			<td style="width:139px;">14025-076</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Name</td>
			<td style="width:180px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">For Western Blot Analysis</td>
			<td style="width:180px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">Mouse anti-Caveolin 1</td>
			<td style="width:180px;">BD Transduction Laboratories</td>
			<td style="width:139px;">610406</td>
			<td style="width:279px;">Western Blot - 1:10000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Tween-20</td>
			<td style="width:180px;">BioRad</td>
			<td style="width:139px;">170-6531</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Nitrocellulose Membrane (0.2 &#956;m)</td>
			<td style="width:180px;">BioRad</td>
			<td>162-0112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Western Lightning Plus-ECL</td>
			<td style="width:180px;">PerkinElmer</td>
			<td style="width:139px;">NEL104001EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">BioRad DC Protein Assay</td>
			<td style="width:180px;">BioRad</td>
			<td style="width:139px;">500-0116</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Sodium dodecyl sulfate (SDS)</td>
			<td style="width:180px;">BioRad</td>
			<td style="width:139px;">161-0302</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Sodium deoxycholate monohydrate (DOC)</td>
			<td style="width:180px;">Sigma</td>
			<td style="width:139px;">D5670</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">IGEPAL CA-630 (NP40)</td>
			<td style="width:180px;">Sigma</td>
			<td style="width:139px;">I8896</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">30% Acrylamide/Bis Solution, 37.5:1</td>
			<td style="width:180px;">BioRad</td>
			<td style="width:139px;">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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10.1K 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

Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, in the case that the KD of the protein of interest has a lethal effect on cells or the anticipated effect of the KD is time-dependent, unconditional KD methods are not appropriate. Conditional systems are more suitable in these cases and have been the subject of much interest. These include Ecdysone-inducible overexpression systems, Cytochrome P-450 induction system1, and the tetracycline regulated gene expression systems. 
The tetracycline regulated gene expression system enables reversible control over protein expression by induction of shRNA expression in the presence of tetracycline. In this protocol, we present an experimental design using functional Tet-ON system in human cancer cell lines for conditional regulation of gene expression. We then demonstrate the use of this system in the study of tumor cell-monocyte interaction.

This protocol serves as a scheme for setting up a functional Tet-ON system in cancer cell lines and its subsequent use, in particular for studying the role of tumor cell-derived proteins in recruitment of monocytes/macrophages to the tumor microenvironment.

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, ...

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissues are widely used for genetic testing. However, FFPE DNA is generally damaged and fragmented during the fixation process with formalin. Therefore, FFPE DNA is sometimes not adequate for genetic testing because of low quality and quantity of DNA. Here we present a method of touch imprint cytology (TIC) to obtain genomic DNA from cancer cells, which can be observed under a microscope. Cell morphology and cancer cell numbers can be evaluated using TIC specimens. Furthermore, the extraction of genomic DNA from TIC samples can be completed within two days. The total amount and quality of TIC DNA obtained using this method was higher than that of FFPE DNA. This rapid and simple method allows researchers to obtain high-quality DNA for genetic testing (e.g., next generation sequencing analysis, digital PCR, and quantitative real time PCR) and to shorten the turnaround time for reporting results.

Obtaining high-quality genomic DNA from tumor tissues is an essential first step for analyzing genetic alterations using next generation sequencing. In this article, we present a simple and rapid method to enrich tumor cells and obtain intact DNA from touch imprint cytology specimens.

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

Measuring Bone Remodeling and Recreating the Tumor-Bone Microenvironment Using Calvaria Co-culture and Histomorphometry

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength and flexibility and allows it to fulfill its functions. Bone is continuously exposed to a variety of stimuli, which in pathological conditions can deregulate bone remodeling. To study bone biology and diseases and evaluate potential therapeutic agents, it has been necessary to develop in vitro and in vivo models.
This manuscript describes the dissection process and culture conditions of calvarias isolated from neonatal mice to study bone formation and the bone tumor microenvironment. In contrast to in vitro and in vivo models, this ex vivo model allows preservation of the three-dimensional environment of the tissue as well as the cellular diversity of the bone while culturing under defined conditions to simulate the desired microenvironment. Therefore, it is possible to investigate bone remodeling and its mechanisms, as well as the interactions with other cell types, such as the interactions between cancer cells and bone.
The assays reported here use calvarias from 5-7 day old BALB/C mice. The hemi-calvarias obtained are cultured in the presence of insulin, breast cancer cells (MDA-MB-231), or conditioned medium from breast cancer cell cultures. After analysis, it was established that insulin induced new bone formation, while cancer cells and their conditioned medium induced bone resorption. The calvarial model has been successfully used in basic and applied research to study bone development and cancer-induced bone diseases. Overall, it is an excellent option for an easy, informative, and low-cost assay.

The ex vivo culture of bone explants can be a valuable tool for the study of bone physiology and the potential evaluation of drugs in bone remodeling and bone diseases. The presented protocol describes the preparation and culture of calvarias isolated from newborn mice skulls, as well as its applications.

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

Analysis of Side Population in Solid Tumor Cell Lines

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great significance for tumor research. Currently, several techniques are used for the identification and purification of CSCs from tumor tissues and tumor cell lines. Separation and analysis of side population (SP) cells are two of the commonly used methods. The methods rely on the ability of CSCs to rapidly expel fluorescent dyes, such as Hoechst 33342. The efflux of the dye is associated with the ATP-binding cassette (ABC) transporters and can be inhibited by ABC transporter inhibitors. Methods for staining cultured tumor cells with Hoechst 33342 and analyzing the proportion of their SP cells by flow cytometry are described. This assay is convenient, fast, and cost-effective. Data generated in this assay can contribute to a better understanding of the effect of genes or other extracellular and intracellular signals on the stemness properties of tumor cells.

A convenient, fast, and cost-effective method to measure the proportion of side population cells in solid tumor cell lines is presented.

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great ...

Isolation of Proximal Fluids to Investigate the Tumor Microenvironment of Pancreatic Adenocarcinoma

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables associated to specific pancreatic pathologies to help preoperative differential diagnosis and patient profiling. Pancreatic juice is a relatively unexplored body fluid, which, due to its close proximity to the tumor site, reflects changes in the surrounding tissue. Here we describe in detail the intraoperative collection procedure. Unfortunately, translating pancreatic juice collection to murine models of PDAC, to perform mechanistic studies, is technically very challenging. Tumor interstitial fluid (TIF) is the extracellular fluid, outside blood and plasma, which bathes tumor and stromal cells. Similarly to pancreatic juice, for its property to collect and concentrate molecules that are found diluted in plasma, TIF can be exploited as an indicator of microenvironmental alterations and as a valuable source of disease-associated biomarkers. Since TIF is not readily accessible, various techniques have been proposed for its isolation. We describe here two simple and technically undemanding methods for its isolation: tissue centrifugation and tissue elution.

Pancreatic juice is a precious source of biomarkers for human pancreatic cancer. We describe here a method for intraoperative collection procedure. To overcome the challenge of adopting this procedure in murine models, we suggest an alternative sample, tumor interstitial fluid, and describe here two protocols for its isolation.

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables ...

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor microenvironment is an important step toward understanding mechanisms that regulate tumor progression. While this can be accomplished through current intravital imaging techniques, it remains challenging due to the heterogeneous nature of tissues and the need for spatial context within the experimental observation. To this end, we have developed an intravital imaging workflow that pairs collagen second harmonic generation imaging, endogenous fluorescence from the metabolic co-factor NAD(P)H, and fluorescence lifetime imaging microscopy (FLIM) as a means to non-invasively compartmentalize the tumor microenvironment into basic domains of the tumor nest, the surrounding stroma or ECM, and the vasculature. This non-invasive protocol details the step-by-step process ranging from the acquisition of time-lapse images of mammary tumor models to post-processing analysis and image segmentation. The primary advantage of this workflow is that it exploits metabolic signatures to contextualize the dynamically changing live tumor microenvironment without the use of exogenous fluorescent labels, making it advantageous for human patient-derived xenograft (PDX) models and future clinical use where extrinsic fluorophores are not readily applicable.

The intravital imaging method described here utilizes collagen second harmonic generation and endogenous fluorescence from the metabolic co-factor NAD(P)H to non-invasively segment an unlabeled tumor microenvironment into tumor, stromal, and vascular compartments for in-depth analysis of 4D intravital images.

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor ...

Industrialized, Artificial Intelligence-guided Laser Microdissection for Microscaled Proteomic Analysis of the Tumor Microenvironment

The tumor microenvironment (TME) represents a complex ecosystem comprised of dozens of distinct cell types, including tumor, stroma, and immune cell populations. To characterize proteome-level variation and tumor heterogeneity at scale, high-throughput methods are needed to selectively isolate discrete cellular populations in solid tumor malignancies. This protocol describes a high-throughput workflow, enabled by artificial intelligence (AI), that segments images of hematoxylin and eosin (H&#38;E)-stained, thin tissue sections into pathology-confirmed regions of interest for selective harvest of histology-resolved cell populations using laser microdissection (LMD). This strategy includes a novel algorithm enabling the transfer of regions denoting cell populations of interest, annotated using digital image software, directly to laser microscopes, thus enabling more facile collections. Successful implementation of this workflow was performed, demonstrating the utility of this harmonized method to selectively harvest tumor cell populations from the TME for quantitative, multiplexed proteomic analysis by high-resolution mass spectrometry. This strategy fully integrates with routine histopathology review, leveraging digital image analysis to support enrichment of cellular populations of interest and is fully generalizable, enabling harmonized harvests of cell populations from the TME for multiomic analyses.

This protocol describes a high-throughput workflow for artificial intelligence-driven segmentation of pathology-confirmed regions of interest from stained, thin tissue section images for enrichment of histology-resolved cell populations using laser microdissection. This strategy includes a novel algorithm enabling the transfer of demarcations denoting cell populations of interest directly to laser microscopes.

The tumor microenvironment (TME) represents a complex ecosystem comprised of dozens of distinct cell types, including tumor, stroma, and immune cell ...