This work is supported by NIH-funded AIDS and Cancer Specimen Resource (<a href="https://www.acsr1.com/">ACSR</a>, UM1 CA181255-2) under its biospecimen science program. The video was filmed and post-production editing was performed by Mayo Clinic Media Services.

All samples were collected and used in compliance with approved Mayo Clinic IRB protocols (PR16-000507 and PR2207-02).
1. Sample preparation
<ol>
	<li>Turn on the tissue floatation water bath. Set the temperature to 39 &#176;C and allow the water to come to temperature. Soak wooden collection sticks in the water bath.</li>
	<li>Identify and retrieve the FFPE tissue blocks to be sectioned.</li>
	<li>Pre-label microscope slides using a histology grade permanent marker that can withstand solvent washes.</li>
	<li>Use a microtome to section the FFPE blocks. Cut at least 2 full-face sections at a thickness of 4-5 &#956;m per section for each block (Figure 1A).</li>
	<li>Transfer the freshly cut ribbon of tissue sections to the pre-warmed tissue floatation bath for slide mounting (Figure 1B, C). 
	NOTE: The warm water helps to &#34;iron&#34; out the wrinkles in the sections (Figure 1C).</li>
	<li>Handling each section sequentially, use forceps to break a single section away from the ribbon (Figure 1D).</li>
	<li>Collect the single section on a pre-labeled microscope slide.
	<ol>
		<li>Submerge the microscope slide at an angle underneath the section, positioning the slide such that the edge of the tissue section touches the slide (Figure 1E).</li>
		<li>Once in contact with the tissue section, pull the slide out of the floatation bath slowly to allow the tissue section to straighten out flush against the slide as it emerges from the water (Figure 1F).</li>
	</ol>
	</li>
	<li>Mount 1 tissue section per slide and repeat until all the sections have been collected.</li>
	<li>Allow the slide-mounted tissue sections to dry thoroughly at room temperature (RT).</li>
</ol>
2. Pathological review
<ol>
	<li>Perform H&#38;E staining on one representative tissue section for each block9.</li>
	<li>Submit the freshly stained H&#38;Es for pathological review by a board-certified pathologist. 
	&#8203;NOTE: During the review, the pathologist determines and records the percentage tumor content in each tissue and circles the tumor area on each H&#38;E slide (see Figure 2 and Figure 3, Row 1). Table 1 outlines the percentage tumor content by cellularity determined during the pathological review of the H&#38;Es for samples A-E shown in Figure 3, Row 1. Sections with &#60;60% tumor content require macro-dissection7. The number of sections needed for nucleic acid extraction depends on the size of the circled tumor area. If insufficient sections were cut in section 1 of the protocol and further cuts are possible, then additional sections may need to be cut.</li>
</ol>
3. Deparaffinization
<ol>
	<li>In a fume hood, prefill two glass staining dishes with undiluted histology grade d-Limonene or a d-Limonene based solvent and 1 glass staining dish with undiluted 200-proof molecular grade ethanol. 
	CAUTION: Avoid d-Limonene contact with skin and eyes, avoid inhalation of vapor or mist, and keep away from sources of ignition. Keep ethanol away from heat, sparks, and open flames, avoid spilling and contact with the skin or eyes, ventilate well and avoid breathing vapors. 
	NOTE: Fill the dishes sufficiently to submerge the racked slides (Figure 4A); 250 mL is required to fill the 20-slide staining dishes shown. Replace the d-Limonene and ethanol washes after every 40 slides. D-Limonene (C10H16) is an alternative, similarly effective, and less toxic dewaxing agent to xylene that is becoming more commonplace in histological methodologies and yields good quality nucleic acid post extraction10,11,12,13,14. Although this protocol may be performed using more biofriendly alternatives15, their effects, if any, on extracted nucleic acid quality remains to be determined.</li>
	<li>Rack the unstained FFPE tissue mounted slides into the glass Coplin slide-racks (Figure 4A, inset).</li>
	<li>Submerge the racked slides in d-Limonene wash 1 for 2 min; gently agitate for the first 20 s. 
	NOTE: To minimize carryover between washes, when removing the rack of slides from a wash, allow the rack to drain briefly before gently dabbing the bottom of the rack on tissue paper to remove excess wash.</li>
	<li>Submerge the racked slides in undiluted D-Limonene wash 2 for 2 min; gently agitate for the first 20 s. Remove, drain and dab the rack again.</li>
	<li>Submerge the racked slides in the ethanol wash for 2 min; gently agitate for the first 20 s. Remove the rack and place it on an absorbent tissue to drain. Allow the slides to air dry for at least 10 min, but no more than 2 h.</li>
</ol>
3. Macrodissection
<ol>
	<li>On the bench, prefill a Coplin glass jar with 50 mL of 3% glycerol in DNase/RNase free water 
	NOTE: Replace the glycerol wash after every 40 slides.</li>
	<li>Pre-label and prefill 1.5 mL microtubes with 160 &#181;L of tissue digestion buffer per microtube. 
	NOTE: Nucleic acid extractions performed after macrodissection used a DNA/RNA FFPE extraction kit (Table of Materials). Thus, the tissue digestion buffer used in this protocol comprised 10 &#181;L of Proteinase K and 150 &#181;L of PKD buffer.</li>
	<li>Trace the pathological markings on the H&#38;E onto the back of the deparaffinized tissue slides. 
	NOTE: Compared to non-deparaffinized tissues (Figure 3, Row 2 and Figure 4B), deparaffinized tissues are white and highly visible (Figure 3, Row 3, and Figure 4C). It is this heightened visibility and discernability of deparaffinized tissue features that permit macrodissection. Place the H&#38;E face down on the bench and place the front of the deparaffinized slide against the back of the matched pathologist reviewed H&#38;E (Figure 4D). Align the deparaffinized tissue with the H&#38;E tissue (Figure 4E). Tracing the pathologist&#39;s markings is a critical step in the macrodissection process, and care should be taken to reproduce these markings as accurately as possible. This can be particularly challenging for small and or disconnected tissues such as samples B, D, and E (Figure 3, Figure 4E, and Figure 4E inset i). To aid tracing, one should use an inky fine or ultrafine nibbed marker (Figure 4E, inset ii) to trace the pathologist-drawn markings. Ethanol wipes may be useful to remove errors and permit retracing if needed.</li>
	<li>Turn the now marked deparaffinized slide tissue face-up and trace the line of the marker with the corner of a clean razor blade to pre-cut the edges of the tumor area.</li>
	<li>Handling each slide sequentially, dip the deparaffinized slides into the 3% glycerol solution. Ensure the tissue is completely submerged before removing the slide slowly (Figure 4F). 
	NOTE: The purpose of the glycerol dip is to dampen the tissue to aid tissue collection but also to reduce the build-up of static charge that can cause repulsion between the tissue and the plastic microtube in which the collected tissue must be placed.</li>
	<li>Gently wipe the back of the slide with a tissue to remove the excess glycerol solution, and lay the slide on the bench, wiped side face down. Allow tissues to briefly airdry for 1-2 min. 
	NOTE: Carry over of excess glycerol into the extraction process can negatively affect the yield and quality of nucleic acid extractions. Tissues should be slightly damp but not visibly wet when collected.</li>
	<li>Depending on where the tumor area of interest is situated on the slide, use the flat edge of the razor blade to either (a) directly collect the tumor tissue, using the razor to scrape/collect the tissue of interest off the slide, or (b) remove and discard non-tumor tissue first before collecting the tumor tissue of interest (Figure 3). 
	NOTE: Collected tissue tends to gather or roll up at the bottom of the blade (Figure 4G)</li>
	<li>Use a wooden stick to remove the collected tissue from the blade (Figure 4H and inset) and transfer it to the appropriate pre-labeled and prefilled microtube (Figure 4I). 
	NOTE: The digestion buffer serves to &#34;pull&#34; the tissue from the wooden pick into the liquid.</li>
	<li>Proceed to nucleic acid extraction. 
	NOTE: Nucleic acid extractions were completed using the DNA/RNA FFPE extraction kit per the manufacturer&#39;s instructions, and the resulting nucleic acids were quantified using a UV-vis spectrophotometer. The resulting RNA was run on the digital gene expression profiling-based DLBCL90 assay16.</li>
</ol>

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The authors have nothing to disclose.

FFPE tissues are frequently heterogeneous admixtures of tumor and non-tumor tissues. High sensitivity genomic tests are becoming increasingly prevalent in both clinical and research settings but can be confounded by the presence of contaminating non-tumor tissue. Indeed, a minimum tumor content of 60% is frequently recommended for genomic studies. Percentage tumor can be determined by the area of tissue occupied by the tumor material or by the proportion of tumor cells within the tissue. Although tumor by area is a commonly used metric for tumor purity, it does not always portray an accurate description of the tissue. Consider two tissues, both with 1000 cells, of which 500 are tumor cells. In tissue A, the 500 non-tumor cells are stromal cells with similar volumes to that of the tumor cell. In this tissue, the percentage of tumor can be considered 50% by both cellularity and area. In tissue B, the 500 non-tumor cells are fat cells with volumes that are 4x that of the tumor cell. In this tissue, the percentage tumor is still 50% by cellularity but 20% by area. A third tissue, tissue C, is composed of 500 tumor cells plus 400 fat cells and 800 stromal cells with volumes that are 4x and 0.5x that of the tumor cells, respectively. Given 100 fat cells equals the volume of 800 stromal cells, the percentage tumor of tissue C is 29% by cellularity (500/1700) but still 20% by area. Tissue D is also comprised of the tumor, fat, and stromal cells with volume ratios of 1x, 4x, and 0.1x. However, the number of cells is 400, 10, and 720, respectively. Thus, the percentage tumor of tissue D is 35% by cellularity (400/1130) but 78% by area. These examples are overly simplistic and do not reflect real-world tissue compositions but clearly convey the importance of tissue composition and the difference between tumor content by area and by cellularity. Importantly, when it comes to enriching tumor content for downstream nucleic acid extraction, tumor cellularity is the more important attribute due to the heightened confounding potential of extracting genomic material from more non-tumor cells than tumor cells. This not only underscores the need to assess the tumor content of tissues in terms of percentage cellularity but also the need to excise unwanted tissue in order to minimize any potential negative effects of the non-tumor tissue. There are several methods available for tissue enrichment, with the main ones being macrodissection and microdissection.
Macrodissection, the method described in this protocol, is relatively quick, simple, and does not require costly or specialized equipment. Although macrodissection can greatly improve tumor content, it is important to understand that it does not completely eliminate non-tumor material. The purpose of macrodissection is to enrich the tissue of interest sufficiently through the exclusion of unwanted tissue in order to reduce the "noise" stemming from unwanted tissue, which in turn can enhance the signal of interest from the tissue of interest. Thus, macrodissection mediated tumor enrichment is a way to enhance the signal-to-noise ratio in order to better detect markers of interest, particularly tumor-specific molecular markers with low abundance or poor expression. However, macrodissection has limitations due to the lack of precision offered by coarse tools such as razor blades and is susceptible to precision issues stemming from the line thickness of the pathologist's marker, as well as potential errors when tracing the pathologists H&amp;E demarcations. As alluded above, it is not possible to achieve 100% tumor purity due to the presence of inherent and tumor-inducing stromal elements (i.e., connective tissue, stromal fibroblasts, blood vessels, benign reactive lymphocytes, macrophages) embedded within the tumor itself. Indeed, many invasive or diffusely infiltrative malignancies induce a robust desmoplastic stromal response, resulting in clusters of tumor cells that are intimately admixed with stromal fibroblasts and other non-neoplastic cell types; where tumors associated with this stromal reaction pattern, such as pancreatic cancer tissues21, may benefit more from digitally guided microdissection rather than manual macrodissection. 
Manual microdissection is performed under a microscope to aid identification, dissection, and isolation of tissues specific cells or populations using a needle or scalpel and has the advantage of increased precision over macrodissection22. However, manual microdissection is a laborious process that lacks the finesse needed for complex tissues with low tumor contents or intricate features that are incompatible with manual dissection. Such tissues can be dissected using high-precision automated methods like laser capture microdissection. Indeed, digitally guided microdissection has been shown to yield higher percentage tumor contents compared to manual macrodissection in pancreatic cancer tissues23. However, the drawbacks of these high precision automated methods, such as the need for specialized, expensive equipment and highly trained individuals, have hindered its incorporation into workflows. A study by de Bruin et al. comparing the effects of macrodissection and laser capture microdissection (LCM) on gene expression profiling found that LCM samples had low total RNA yields (30 ng average) and required two rounds of mRNA amplification in order to meet the cDNA library prep input threshold24. The authors found that the resulting LCM gene expression profiles were affected by the rounds of mRNA amplification more than macrodissected profiles were affected by non-tumor stromal contributions and concluded that macrodissection could be adequately used to generate reliable gene expression data24. 
A significant advantage of NanoString digital gene expression profiling, particularly when working with highly degraded FFPE derived RNA, is that it does not require enzymatic dependent processes such as RNA amplification or preparation of cDNA libraries. However, assays are typically optimized for inputs between 50-300 ng of total RNA25,26, which, based on the findings of de Bruin et al.24, may not be compatible with microdissected tissues without increasing the tissue input; an unfavorable demand in an era where tissue samples are increasingly collected as small biopsies rather than surgical resections. The RNA inputs used for the DLBCL90 assay ranged from 68.5-300 ng for both the macrodissected and non-dissected tissues. The results show that macrodissection resulted in call changes in 60% of the samples examined and that these changes were observed irrespective of RNA input of the macrodissected samples. However, the COO probability for the low RNA input did encroach the COO GCB/UNC probability call threshold, where the thresholds are 0 to &lt;0.1 for GCB, 0.1-0.9 for UNC, and &gt;0.9 to 1.0 for ABC calls20. The principal DLBCL COO subtypes are GCB and ABC, which make up 41% and 44% of all DLBCL cases, with UNC representing an intermediate group of the two and ABC being the most aggressive20,27. Thus, while the COO call change upon macrodissection of sample C did not cause a frank change in COO subtype from GCB to ABC, the change from GCB to UNC may suggest a shift towards a more aggressive disease. Moreover, recent studies indicate that the UNC subtype is not simply just an intermediate subtype and that it may potentially possess subtype-specific therapeutically exploitable attributes28. Similarly, macrodissection of samples A and E did not cause frank changes in DHITsig calls from DH negative to DH positive, or vice versa. However, the movements of a GCB sample (sample A) from NEG to UNCLASS and an ABC sample (sample E) from UNCLASS to NEG upon macrodissection are biologically appropriate as double hit translocations involving BCL2 are reported to be an exclusively GCB phenomenon19. Although translocations are traditionally and ubiquitously detected by FISH in clinical settings, there is a growing momentum to identify an alternative less involved and time-consuming method for their detection. The DLBCL90 assay is an important tool that addresses this need, where the rationale for its use is strengthened by the finding that this assay is capable of detecting translocations cryptic to FISH probes used in clinical diagnostics29. 
The macrodissection protocol described above outlines a simple method that enables researchers to increase the tumor content of tissue samples that would ordinarily fall below commonly used study inclusion criteria thresholds. Including macrodissection in a study workflow enables researchers to salvage poorly tumor-dense tissues from study exclusion by increasing their tumor content. In turn, this permits increased confidence that the resulting RNA and DNA eluates represent the tumor under genomic investigation. Although other more precise methods for tissue dissection do exist, for tumors that grow in a more expansive, non-infiltrative, sheet-like, or solid fashion, macrodissection is likely sufficient. The results presented here highlight the importance of tumor purity in genomic assays and macrodissection as a reliable tool to achieve this.

Formalin-fixed paraffin-embedded (FFPE) tissues, collected as part of the normal clinical diagnostic process and retained in clinical tissue repositories, represent a vast resource for human research, including cancer research1. As our understanding of human disease deepens, it is becoming increasingly clear that diseases, previously thought to be single entities based on morphological and immunophenotypical characteristics, are in fact comprised of distinct molecular subtypes that require molecular subtyping assays. Consequently, high sensitivity genomic assays capable of discerning these subtypes have become increasingly important2. Although FFPE tissues are renowned for being poorly compatible with genomic techniques due to fixation-related issues, as technology and protocols evolve, these techniques are becoming increasingly compatible with this clinically ubiquitous tissue format3,4,5. However, FFPE tissues are often admixtures of tumor and non-tumor tissue materials, where the presence of non-tumor material is frequently unwanted and can, if present at a high proportion, significantly undermine and impact the results of genomic analyses6. Indeed, a minimum tumor content of 60% is frequently used for such analyses, where tissues that fall short of this threshold can be excluded, despite otherwise fulfilling the study criteria7. This can be particularly problematic in rare disease settings, where patient tissues are precious and difficult to collect in high numbers.
Macrodissection is a method that minimizes the effects of low tumor content by reducing the amount of normal tissue3. The removal of such confounding non-tumor material prior to nucleic acid extraction can significantly augment the tumor percentage content and thus the tumor purity of the extracted nucleic acids. Tissue resection critically relies upon expert pathological review, wherein the tumor region is identified and circled on a freshly generated hematoxylin and eosin (H&#38;E) stained tissue section by a board-certified pathologist8. The circled H&#38;E is then used to guide the removal and collection of unwanted and target tissues, respectively. This protocol describes the steps of macrodissection from pathological review through tissue harvesting as performed at the AIDS and Cancer Specimen Resource (ACSR) Technical Core Laboratory at the Mayo Clinic.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>200-proof ethanol</td>
			<td>Decon</td>
			<td>2701</td>
			<td></td>
		</tr>
		<tr>
			<td>AllPrep DNA/RNA FFPE Kit</td>
			<td>Qiagen</td>
			<td>80234</td>
			<td>DNA/RNA FFPE extraction kit</td>
		</tr>
		<tr>
			<td>Coplin pots</td>
			<td>Various</td>
			<td>x</td>
			<td></td>
		</tr>
		<tr>
			<td>DLBCL90 probes</td>
			<td>NanoString</td>
			<td>various</td>
			<td>Digital gene expression profiling based DLBCL90 assay</td>
		</tr>
		<tr>
			<td>d-Limonene</td>
			<td>VWR</td>
			<td>89376-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Various</td>
			<td>x</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass micrscope slides</td>
			<td>FisherBrand</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>VWR</td>
			<td>0854-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Master kits</td>
			<td>NanoString</td>
			<td>various</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica</td>
			<td>RM2265</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtubes</td>
			<td>Ambion</td>
			<td>AM12400</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop One</td>
			<td>Thermo Scientific</td>
			<td>ND-ONE-W</td>
			<td>Spectrophotometer for DNA, RNA and protein qualitation</td>
		</tr>
		<tr>
			<td>nCounter</td>
			<td>NanoString</td>
			<td>x</td>
			<td>Digital gene expression profiling platform used to run the DLBCL90 assay</td>
		</tr>
		<tr>
			<td>Permanent marker</td>
			<td>Electrib Microscope Sciences</td>
			<td>72109-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blade dispenser</td>
			<td>Electrib Microscope Sciences</td>
			<td>71985-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blades</td>
			<td>Electrib Microscope Sciences</td>
			<td>71985-23</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue digestion buffer</td>
			<td>Qiagen</td>
			<td>80234</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure water</td>
			<td>VWR</td>
			<td>SH30538.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Waterbath</td>
			<td>Triangle Biomedical Sciences</td>
			<td>TFB-120</td>
			<td></td>
		</tr>
		<tr>
			<td>Wooden stick</td>
			<td>FisherBrand</td>
			<td>22363158</td>
			<td></td>
		</tr>
	</tbody>
</table>

enhancing tumor content through tumor macrodissection

The presence of contaminating non-tumor tissues in formalin-fixed paraffin-embedded (FFPE) tissues can greatly undermine genomic studies. Herein we describe macrodissection, a method designed to augment the percentage tumor content of a tissue specimen by removing and eliminating unwanted tissue prior to performing downstream nucleic acid extractions. FFPE tissue blocks were sectioned to produce 4-5 &#181;m slide-mounted tissue sections. A representative section was submitted for hematoxylin and eosin (H&#38;E) staining and subsequently reviewed by a board-certified pathologist. During the review, the pathologist identified and marked the regions of tumor tissue in the H&#38;E. Once complete, the demarked H&#38;E was used to guide resection of the serial unstained sections from the same tissue block. To demonstrate the effects of macrodissection, RNA extracted from matched macrodissected and non-dissected Diffuse Large B-Cell Lymphomas (DLBCL) were run on a digital gene expression assay capable of determining DLBCL subtype and BCL2 translocation status. The results showed that macrodissection changed the subtype or BCL2 translocation status calls in 60% of the samples examined. In conclusion, macrodissection is a simple and effective method for performing tumor enrichment prior to nucleic acid extractions, the product of which can then be confidently used in downstream genomic studies.

A total of 5 Diffuse Large B-Cell Lymphoma (DLBCL) FFPE tissue blocks were sectioned, and the resulting sections were either macrodissected or not prior to nucleic acid extraction. The extracted RNA was run on the DLBCL90 assay16. Macrodissected samples were run twice, once using 5 &#181;L of RNA stock concentration but no more than 300 ng of total RNA input and once using 5 &#181;L of RNA stock diluted to match the RNA inputs of their respective non-dissected counterparts. The DLBCL90 results are outlined in Table 2.
DLBCL is comprised of 3 distinct cell of origin (COO) subtypes with different therapeutic responsiveness, namely GCB, ABC, and an intermediate group known as unclassified or UNC17,18. Translocations involving MYC, BCL2, and or BCL6 alone or in combination (double or triple hit) are also frequently observed in DLBCL, particularly in the GCB subtype19. The DLBCL90 assay is an expansion of its predecessor, the Lymph2Cx clinical assay, and is thus capable of DLBCL COO subtype determination but was principally developed to identify samples harboring double hit (DH) translocations involving BCL2 using digital gene expression as an alternative to fluorescence in-situ hybridization (FISH)16,20. The results in Table 2 show that macrodissection changed either the COO or the DHITsig status calls for 60% (3/5) of the samples examined.
Macrodissection of sample A had no effect on the COO call but changed the DHITsig call from NEG to UNCLASS, and this change was observed irrespective of the macrodissected sample RNA input and with similar probability scores (0.224, 0.254). In contrast, macrodissection of sample C had no effect on the DHITsig call but changed the COO call from GCB to UNC. Again, this change was observed irrespective of macrodissected sample RNA input. However, at 0.117, the COO call probability was closer to the call threshold of 0.1 for the macrodissected sample with reduced RNA input. Similar to sample A, macrodissection of sample E had no effect on the COO call but changed the DHITsig call. However, for sample E the call changed from UNCLASS to NEG and did so irrespective of the macrodissected sample RNA input with reasonably similar probability calls (0.849, 0.833). Notably, this call change to DHITsig NEG makes biological sense given that sample E was found to ABC-DLBCL, and double hit translocations involving BCL2 have been reported to be exclusively observed in GCB-DLBCL19.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62961/62961fig01.jpg" /> 
Figure 1: Generating slide-mounted tissue sections using a microtome. (A) FFPE tissue block held in place by the microtome chuck and cut to produce a ribbon of sequential FFPE tissue sections. (B) Using pre-soaked wooden sticks, the ribbon is collected from the microtome and transferred to a warm water bath. (C) The warmth of the water helps to iron out the creases in the tissue ribbon. (D) Individual FFPE tissue sections are removed from the tissue ribbon by placing closed forceps at the junction of two sections and gently opening the forceps, which breaks sections away from one another. (E) Sections are collected from the water by submerging a glass slide at an angle and gently moving the side towards the tissue section until the edge of the section touches the glass slide. (F) Once the slide and section are touching, slowly remove the slide from the water, allowing the tissue section to fall flush against the slide. <a href="https://www.jove.com/files/ftp_upload/62961/62961fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62961/62961fig02.jpg" /> 
Figure 2: Pathology and histological review of a hematoxylin and eosin (H&#38;E) stained sections. (A,B) The H&#38;E tissue section is subjected to microscopic review by a board-certified pathologist. (C,D) Once the pathologist has viewed the entire tissue and found that it is not 100% tumor tissue, the pathologist will use a marker to encircle the tumor area of the tissue. (E,F) Holding the marked H&#38;E against the original FFPE tissue block shows that not all the tissue is tumor material. <a href="https://www.jove.com/files/ftp_upload/62961/62961fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62961/62961fig03.jpg" /> 
Figure 3: Tissue samples. This image demonstrates the pathologically reviewed and tumor marked H&#38;Es (Row 1), unprocessed slide-mounted FFPE tissue sections (Row 2), deparaffinized slide-mounted FFPE tissue sections (Row 3), deparaffinized slide-mounted FFPE tissue sections with pathology markings traced on the back of the slide (Row 4), deparaffinized and macrodissected slide-mounted FFPE tissue sections (Row 5) for the 5 samples (A-E) used to demonstrate this protocol. <a href="https://www.jove.com/files/ftp_upload/62961/62961fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62961/62961fig04.jpg" /> 
Figure 4: Deparaffinization and macro-dissection of FFPE tissue sections: (A) In a fume hood, FFPE tissues mounted in a slide rack are washed in two d-limonene washes and one ethanol wash. (B,C) Post wash, all the paraffin has been removed, and only the tissue remains on the slide, which is now white and highly visible compared to its prewashed counterpart. (D) The deparaffinized slide-mounted tissue section is placed face down on the back of its matching marked H&#38;E. (E) The tumor area markings on the H&#38;E are then traced on the back of the deparaffinized slide using a fine or ultrafine nibbed permanent marker (F) The marked deparaffinized slide-mounted tissue section is then dipped in a glycerol wash to dampen the tissue section for collection. The slide is removed slowly from the glycerol, and the back of the slide wiped with a tissue to remove excess glycerol before laying the slide tissue face-up on the bench. (G) Using the flat side of a clean razor blade, the tissue is macrodissected, and the unwanted tissue outside of the pathologist markings is discarded before collecting the tissue of interest, which gathers along the edge of the blade. (H) A wooden stick is used to remove the collected tissue from the edge of the blade. (I) The tissue is then transferred to a pre-labeled microtube prefilled tissue digestion buffer and is ready for nucleic acid extraction. <a href="https://www.jove.com/files/ftp_upload/62961/62961fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:font-size="7px" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2" style="min-width: 80px;">Sample ID</td>
			<td colspan="2" style="min-width: 80px;">Whole Tissue (a)</td>
			<td colspan="2" style="min-width: 80px;">Circled area of tissue (b)</td>
			<td rowspan="2" style="min-width: 80px;">Overall tumor content of whole tissue (c)</td>
			<td rowspan="2" style="min-width: 80px;">Fold increase in tumor content by macrodissection (d)</td>
			<td colspan="2" style="min-width: 80px;">Not macrodissected (e)</td>
			<td colspan="2" style="min-width: 80px;">Macrodissected (f)</td>
		</tr>
		<tr>
			<td style="min-width: 80px;">% Viable tumor</td>
			<td style="min-width: 80px;">% Other</td>
			<td style="min-width: 80px;">% Viable tumor</td>
			<td style="min-width: 80px;">% Other</td>
			<td style="min-width: 80px;"># of 5 &#181;m slides extracted</td>
			<td style="min-width: 80px;">RNA conc (ng/&#181;L)</td>
			<td style="min-width: 80px;"># of 5 &#181;m slides extracted</td>
			<td style="min-width: 80px;">RNA conc 
			(ng/&#181;L)</td>
		</tr>
		<tr>
			<td style="min-width: 80px;">Sample A</td>
			<td style="min-width: 80px;">60</td>
			<td style="min-width: 80px;">40</td>
			<td style="min-width: 80px;">75</td>
			<td style="min-width: 80px;">25</td>
			<td style="min-width: 80px;">45</td>
			<td style="min-width: 80px;">1.7</td>
			<td style="min-width: 80px;">2</td>
			<td style="min-width: 80px;">19.0</td>
			<td style="min-width: 80px;">4</td>
			<td style="min-width: 80px;">58.3</td>
		</tr>
		<tr>
			<td style="min-width: 80px;">Sample B</td>
			<td style="min-width: 80px;">60</td>
			<td style="min-width: 80px;">40</td>
			<td style="min-width: 80px;">65</td>
			<td style="min-width: 80px;">35</td>
			<td style="min-width: 80px;">39</td>
			<td style="min-width: 80px;">1.7</td>
			<td style="min-width: 80px;">1</td>
			<td style="min-width: 80px;">34.0</td>
			<td style="min-width: 80px;">2</td>
			<td style="min-width: 80px;">60.0</td>
		</tr>
		<tr>
			<td style="min-width: 80px;">Sample C</td>
			<td style="min-width: 80px;">40</td>
			<td style="min-width: 80px;">60</td>
			<td style="min-width: 80px;">65</td>
			<td style="min-width: 80px;">35</td>
			<td style="min-width: 80px;">26</td>
			<td style="min-width: 80px;">2.5</td>
			<td style="min-width: 80px;">1</td>
			<td style="min-width: 80px;">13.7</td>
			<td style="min-width: 80px;">2</td>
			<td style="min-width: 80px;">46.2</td>
		</tr>
		<tr>
			<td style="min-width: 80px;">Sample D</td>
			<td style="min-width: 80px;">35</td>
			<td style="min-width: 80px;">65</td>
			<td style="min-width: 80px;">90</td>
			<td style="min-width: 80px;">10</td>
			<td style="min-width: 80px;">32</td>
			<td style="min-width: 80px;">2.9</td>
			<td style="min-width: 80px;">1</td>
			<td style="min-width: 80px;">57.3</td>
			<td style="min-width: 80px;">2</td>
			<td style="min-width: 80px;">60.0</td>
		</tr>
		<tr>
			<td style="min-width: 80px;">Sample E</td>
			<td style="min-width: 80px;">20</td>
			<td style="min-width: 80px;">80</td>
			<td style="min-width: 80px;">30</td>
			<td style="min-width: 80px;">70</td>
			<td style="min-width: 80px;">6</td>
			<td style="min-width: 80px;">5.0</td>
			<td style="min-width: 80px;">3</td>
			<td style="min-width: 80px;">25.2</td>
			<td style="min-width: 80px;">3</td>
			<td style="min-width: 80px;">44.6</td>
		</tr>
	</tbody>
</table>
Table 1: Pathology review data. The table shows (a) the percentage of viable tumor in the whole tissue section by area, (b) the percentage of viable tumor in the area circled/marked by the pathologist during the review by cellularity, (c) the estimated overall tumor cellularity of the whole tissue (a x b), (d) the estimated fold increase in tumor cellularity achieved with macrodissection, (e and f) the number of 5 &#181;m unstained FFPE slide-mounted tissue sections extracted and the resulting RNA concentrations for matched non-macrodissected and macrodissected samples. % Other refers to all other tissues present in a given sample that is not tumor tissue and can include connective tissue, stromal fibroblasts, blood vessels as well as other inherent stromal elements. <a href="https://www.jove.com/files/ftp_upload/62961/Table 1-62961R3.xlsx" target="_blank">Please click here to download this Table.</a>
<table border="1" fo:font-size="8px" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Sample ID</td>
			<td>RNA input (ng)</td>
			<td>DLBCL90 COO call</td>
			<td>DLBCL90 call&#160; probability</td>
			<td>DHITsig call</td>
			<td>DHITsig pos probability</td>
			<td>DHITsig neg probability</td>
		</tr>
		<tr>
			<td rowspan="5">Not macrodissected</td>
			<td>Sample A</td>
			<td>95.0</td>
			<td>GCB</td>
			<td>0.000</td>
			<td>NEG</td>
			<td>0.135</td>
			<td>0.865</td>
		</tr>
		<tr>
			<td>Sample B</td>
			<td>170.0</td>
			<td>GCB</td>
			<td>0.000</td>
			<td>NEG</td>
			<td>0.032</td>
			<td>0.968</td>
		</tr>
		<tr>
			<td>Sample C</td>
			<td>68.5</td>
			<td>GCB</td>
			<td>0.028</td>
			<td>NEG</td>
			<td>0.033</td>
			<td>0.967</td>
		</tr>
		<tr>
			<td>Sample D</td>
			<td>286.5</td>
			<td>ABC</td>
			<td>0.998</td>
			<td>NEG</td>
			<td>0.002</td>
			<td>0.998</td>
		</tr>
		<tr>
			<td>Sample E</td>
			<td>126.0</td>
			<td>ABC</td>
			<td>0.989</td>
			<td>UNCLASS</td>
			<td>0.212</td>
			<td>0.788</td>
		</tr>
		<tr>
			<td rowspan="5">Macrodissected</td>
			<td>Sample A_M</td>
			<td>291.7</td>
			<td>GCB</td>
			<td>0.000</td>
			<td>UNCLASS</td>
			<td>0.224</td>
			<td>0.776</td>
		</tr>
		<tr>
			<td>Sample B_M</td>
			<td>300.0</td>
			<td>GCB</td>
			<td>0.000</td>
			<td>NEG</td>
			<td>0.016</td>
			<td>0.984</td>
		</tr>
		<tr>
			<td>Sample C_M</td>
			<td>231.2</td>
			<td>UNCLASS</td>
			<td>0.210</td>
			<td>NEG</td>
			<td>0.015</td>
			<td>0.985</td>
		</tr>
		<tr>
			<td>Sample D_M</td>
			<td>300.0</td>
			<td>ABC</td>
			<td>0.999</td>
			<td>NEG</td>
			<td>0.002</td>
			<td>0.998</td>
		</tr>
		<tr>
			<td>Sample E_M</td>
			<td>223.2</td>
			<td>ABC</td>
			<td>0.987</td>
			<td>NEG</td>
			<td>0.151</td>
			<td>0.849</td>
		</tr>
		<tr>
			<td rowspan="5">Macrodissected &#38; RNA diluted</td>
			<td>Sample A_M</td>
			<td>95.0</td>
			<td>GCB</td>
			<td>0.000</td>
			<td>UNCLASS</td>
			<td>0.254</td>
			<td>0.746</td>
		</tr>
		<tr>
			<td>Sample B_M</td>
			<td>170.0</td>
			<td>GCB</td>
			<td>0.000</td>
			<td>NEG</td>
			<td>0.023</td>
			<td>0.977</td>
		</tr>
		<tr>
			<td>Sample C_M</td>
			<td>68.5</td>
			<td>UNCLASS</td>
			<td>0.117</td>
			<td>NEG</td>
			<td>0.027</td>
			<td>0.973</td>
		</tr>
		<tr>
			<td>Sample D_M</td>
			<td>286.5</td>
			<td>ABC</td>
			<td>0.999</td>
			<td>NEG</td>
			<td>0.002</td>
			<td>0.998</td>
		</tr>
		<tr>
			<td>Sample E_M</td>
			<td>126.0</td>
			<td>ABC</td>
			<td>0.995</td>
			<td>NEG</td>
			<td>0.167</td>
			<td>0.833</td>
		</tr>
	</tbody>
</table>
Table 2: DLBCL90 digital gene expression assay results. Five samples (A-E) were either not macrodissected or macrodissected before nucleic acid extractions were performed. The resulting RNA was run on the DLBCL90 assay, for which the maximum RNA input volume is 5 &#181;L. Non-macrodissected samples were run using 5 &#181;L of stock RNA. Each macrodissected sample was run twice using (a) 5 &#181;L of stock RNA unless aliquots of 60 ng/&#181;L were possible and (b) 5 &#181;L of stock RNA diluted to match the concentrations/input of their non-macrodissected counterparts. The suffix _M denotes that that sample was macrodissected. <a href="https://www.jove.com/files/ftp_upload/62961/Table 2-62961R3.xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Enhancing Tumor Content through Tumor Macrodissection at JoVE.com

Enhancing Tumor Content through Tumor Macrodissection

This protocol presents a method to increase the percent tumor content of formalin-fixed paraffin-embedded tissue samples.

The presence of contaminating non-tumor tissues in formalin-fixed paraffin-embedded (FFPE) tissues can greatly undermine genomic studies. Herein we ...

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9.5K Views.  Mayo Clinic. This protocol presents a method to increase the percent tumor content of formalin-fixed paraffin-embedded tissue samples. 

Video: Enhancing Tumor Content through Tumor Macrodissection

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration2. In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor3. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.

We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is ...

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

An In vitro FluoroBlok Tumor Invasion Assay

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to both secrete proteases that break down the basement membrane as well as migrate in order to be invasive. BD BioCoat Tumor Invasion System provides cells with conditions that allow assessment of their invasive property in vitro1,2. It consists of a BD Falcon FluoroBlok 24-Multiwell Insert Plate with an 8.0 micron pore size PET membrane that has been uniformly coated with BD Matrigel Matrix. This uniform layer of BD Matrigel Matrix serves as a reconstituted basement membrane in vitro providing a true barrier to non-invasive cells while presenting an appropriate protein structure to study invasion. The coating process occludes the pores of the membrane, blocking non-invasive cells from migrating through the membrane. In contrast, invasive cells are able to detach themselves from and migrate through the coated membrane. Quantitation of cell invasion can be achieved by either pre- or post-cell invasion labeling with a fluorescent dye such as DiIC12(3) or calcein AM, respectively, and measuring the fluorescence of invading cells. Since the BD FluoroBlok membrane effectively blocks the passage of light from 490-700 nm at &gt;99% efficiency, fluorescently-labeled cells that have not invaded are not detected by a bottom-reading fluorescence plate reader. However, cells that have invaded to the underside of the membrane are no longer shielded from the light source and are detected with the respective plate reader. This video demonstrates an endpoint cell invasion assay, using calcein AM to detect invaded cells.

An In vitro FluoroBlok Tumor Invasion Assay

This video demonstrates how to measure cell invasion through cell culture inserts using a fluorescence-based methodology.

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to ...

Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as tumor-propagating cells, or tumor-initiating cells. It is thought that targeting these self-renewing cells for destruction will block tumor progression and stop relapse, greatly improving patient prognosis1. The most common way to determine the frequency of self-renewing cells within a tumor is a limiting dilution cell transplantation assay, in which tumor cells are transplanted into recipient animals at increasing doses; the proportion of animals that develop tumors is used the calculate the number of self-renewing cells within the original tumor sample2, 3. Ideally, a large number of animals would be used in each limiting dilution experiment to accurately determine the frequency of tumor-propagating cells. However, large scale experiments involving mice are costly, and most limiting dilution assays use only 10-15 mice per experiment. 
Zebrafish have gained prominence as a cancer model, in large part due to their ease of genetic manipulation and the economy by which large scale experiments can be performed. Additionally, the cancer types modeled in zebrafish have been found to closely mimic their counterpart human disease4. While it is possible to transplant tumor cells from one fish to another by sub-lethal irradiation of recipient animals, the regeneration of the immune system after 21 days often causes tumor regression5. The recent creation of syngeneic zebrafish has greatly facilitated tumor transplantation studies 6-8. Because these animals are genetically identical, transplanted tumor cells engraft robustly into recipient fish, and tumor growth can be monitored over long periods of time. Syngeneic zebrafish are ideal for limiting dilution transplantation assays in that tumor cells do not have to adapt to growth in a foreign microenvironment, which may underestimate self-renewing cell frequency9, 10. Additionally, one-cell transplants have been successfully completed using syngeneic zebrafish8 and several hundred animals can be easily and economically transplanted at one time, both of which serve to provide a more accurate estimate of self-renewing cell frequency.
Here, a method is presented for creating primary, fluorescently-labeled T-cell acute lymphoblastic leukemia (T-ALL) in syngeneic zebrafish, and transplanting these tumors at limiting dilution into adult fish to determine self-renewing cell frequency. While leukemia is provided as an example, this protocol is suitable to determine the frequency of tumor-propagating cells using any cancer model in the zebrafish.

Limiting dilution cell transplantation assays are used to determine the frequency of tumor-propagating cells. This protocol describes a method for generating syngeneic zebrafish that develop fluorescently-labeled leukemia and details how to isolate and transplant these leukemia cells at limiting dilution into the peritoneal cavity of adult zebrafish.

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as ...

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

A tube formation assay is used to evaluate vascular activity of tumor cells.

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, and multiwell-based assays are used to measure transcription factor activity. However, these assays are nonquantitative, lack specificity, may involve the use of radiolabeled oligonucleotides, and may not be adaptable for the screening of inhibitors of DNA binding. On the other hand, using a quantitative DNA-binding enzyme-linked immunosorbent assay (D-ELISA) assay, we demonstrate nuclear protein interactions with DNA using the RUNX2 transcription factor that depend on specific association with consensus DNA-binding sequences present on biotin-labeled oligonucleotides. Preparation of cells, extraction of nuclear protein, and design of double stranded oligonucleotides are described. Avidin-coated 96-well plates are fixed with alkaline buffer and incubated with nuclear proteins in nucleotide blocking buffer. Following extensive washing of the plates, specific primary antibody and secondary antibody incubations are followed by the addition of horseradish peroxidase substrate and development of the colorimetric reaction. Stop reaction mode or continuous kinetic monitoring were used to quantitatively measure protein interaction with DNA. We discuss appropriate specificity controls, including treatment with non-specific IgG or without protein or primary antibody. Applications of the assay are described including its utility in drug screening and representative positive and negative results are discussed.

We developed a quantitative DNA-binding, ELISA-based assay to measure transcription factor interactions with DNA. High specificity for the RUNX2 protein was achieved with a consensus DNA-recognition oligonucleotide and specific monoclonal antibody. Colorimetric detection with an enzyme-coupled antibody substrate reaction was monitored in real time.

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing

Efforts to detect and investigate key oncogenic mutations have proven valuable to facilitate the appropriate treatment for cancer patients. The establishment of high-throughput, massively parallel "next-generation" sequencing has aided the discovery of many such mutations. To enhance the clinical and translational utility of this technology, platforms must be high-throughput, cost-effective, and compatible with formalin-fixed paraffin embedded (FFPE) tissue samples that may yield small amounts of degraded or damaged DNA. Here, we describe the preparation of barcoded and multiplexed DNA libraries followed by hybridization-based capture of targeted exons for the detection of cancer-associated mutations in fresh frozen and FFPE tumors by massively parallel sequencing. This method enables the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus conferring high sensitivity for detecting low frequency mutations.

We describe the preparation of barcoded DNA libraries and subsequent hybridization-based exon capture for detection of key cancer-associated mutations in clinical tumor specimens by massively parallel "next generation" sequencing. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus yielding high sensitivity for detecting low frequency mutations.

Efforts  to detect and investigate key oncogenic mutations have proven valuable to  facilitate the appropriate treatment for cancer patients. The ...

Monitoring Functionality and Morphology of Vasculature Recruited by Factors Secreted by Fast-growing Tumor-generating Cells

The subcutaneous matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic factors. In this method, desired factors are introduced into cold-liquid ECM-mimic gel which, after subcutaneous injection, solidifies to form an environment mimicking the cancer milieu. This matrix permits the penetration of host cells, such as endothelial cells, and therefore, the formation of vasculature.
Herein we propose a new modified matrigel plug assay, which can be exploited to illustrate the angiogenic potential of a pool of factors secreted by cancer cells, as opposed to a specific factor (e.g., bFGF and VEGF) or agent. The plug containing ECM-mimic gel is utilized to introduce the host (i.e., mouse) with a pool of factors secreted to the C.M. of fast-growing tumor-generating glioblastoma cells. We have previously described an extensive comparison of the angiogenic potential of U-87 MG human glioblastoma and its dormant-derived clone, in this system model, showing induced angiogenesis in the U-87 MG parental cells. The C.M. is prepared by filtering collected media from confluent tissue culture plates of either cell line following 48 hr incubation. Hence, it contains only factors secreted by the cells, without the cells themselves. Described here is the combination of two imaging modalities, microbubbles contrast-enhanced ultrasound imaging and intravital fibered-confocal endomicroscopy, for an accurate, real-time characterization of the extent, morphology and functionality of newly-formed blood vessels within the plugs.

We describe a matrigel plug assay to illustrate angiogenic potential of a pool of factors secreted by cancer cells using two complementary imaging modalities, ultrasound and endomicroscopy. The matrigel, an extracellular matrix (ECM)-mimic gel, is utilized to introduce the host (mouse) with angiogenic factors secreted to the conditioned media (C.M.).&#160;

The subcutaneous matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic factors. In this method, desired ...