Name: non-destructive evaluation of regional cell density within tumor aggregates following drug treatment
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Description: Watch this Scientific Journal Video about Non-Destructive Evaluation of Regional Cell Density Within Tumor Aggregates Following Drug Treatment at JoVE.com

This study was supported by NIH R01 BRG CA207725 (MB/DTC) and NIH R01 CA233188 (MB). We would like to thank AMC Pharmacy for the Trastuzumab provided for these experiments.

AU565 (HER2+) and MDA-MB-231 breast cancer cell lines were used for the present study (see Table of Materials).
1. Preparing tumor aggregates
<ol>
	<li>Prepare AU565 (HER2+) breast cancer cell growth media using Roswell Park Memorial Institute (RPMI) 1640 basal medium (+) in L-glutamine supplemented with 10% (v/v) fetal bovine serum and 1% penicillin/streptomycin (see Table of Materials).</li>
	<li>Prepare MDA-MB-231 triple-negative breast c...

<ol>
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Significance 
Multicellular tumor spheroids (MCTSs) are powerful 3D in vitro models for studying tumor progression and drug screening1,2,3. Advancing the utility of these relatively simple aggregate models relies heavily on the characterization of their key features, such as morphology and cell density, which are known to influence both tumor model progression and therapeutic response. However, the...

Researchers have largely turned to benchtop 3D culture in vitro systems to study some of the key features of tumor progression. Much of this research has been led by the re-emergence of multicellular tumor spheroids (MCTSs) and more complex organoids1,2. Although these models are avascular, they provide a powerful tool for recapitulating physiological and pathological processes that occur in vivo3,4,5. In particular, medium-sized models (300-500 &#181;m diameter) can mimic key tumor features such as 3D structure, pathophysiological gradients, and metastatic signaling due to hypoxia within the core. It is well documented that these models display the characteristic concentric layers seen in vascularized in vivo tumors, namely, an outer layer of proliferative cells, a transitional layer of senescent/quiescent cells, and cells experiencing hypoxia in the core3,6,7,8,9.&#160;Unique insight can be gained from these models by characterizing cell behavior within these layers, during development and in response to the drug. However, the requisite MCTS size, necessary to develop the gradients that make them such powerful in vitro models, drastically limits the tools used for non-destructive assessment. Indeed, one of the biggest challenges with non-destructive analysis of MTCSs is quantifying cell-scale details. Bright-field and phase-contrast microscopy are routinely utilized to assess 3D MCTSs growth and development non-destructively. However, these modalities are limited to 2D projections, lacking the capacity to visualize the crucial 3D structure of these models10,11,12,13. Information on cytotoxicity and cell proliferation is typically collected through fluorescent imaging (i.e., light-sheet microscopy, confocal microscopy) or ex vivo immunohistological staining14,15,16. While these approaches provide valuable, high-resolution information on tissue structure, cellular density, and cellular function, they often require sample preparation such as optical clearing, fixing/staining, or embedding that prevents longitudinal analyses.
Optical Coherence Tomography (OCT) is a non-destructive structural imaging modality that has the potential to overcome some of the challenges mentioned above. It boasts cellular resolution and a sufficiently wide field of view (up to 10 mm x 10 mm) capable of visualizing entire multicellular aggregates17,18,19. Importantly, due to the visible nature of the light used, this technique is completely non-destructive and label-free17. Also, samples can be imaged in situ without requiring sample preparation, such that samples can be taken straight from the incubator, quickly scanned with OCT (scan duration ~5-10 min), then returned to the incubator, enabling longitudinal characterization. Many studies seeking to use OCT to analyze tumor spheroid behavior have recently emerged. In one of the most exciting demonstrations, Huang et al. used OCT to non-destructively detect necrotic cores within large tumor spheroid models, noting that live and dead cell regions possess discernible differences in optical attenuation, which may be utilized for label-free viability monitoring20. Similarly, Hari et al. conducted refractive index (RI) measurements of human colon cancer (HCT116) spheroids imaged with OCT to study the presence of hypoxia within the samples21. Their measurements were not sufficient for direct inferences, though they did observe lower RI in locations that correlated with the site, though not size, of necrotic cores, later identified via confocal microscopy. Abd El-Sadek et al. used OCT to visualize and quantify regional tissue viability of breast cancer tumor models22. They reported two OCT-based methods for visualizing tissue dynamics and showed a moderate correlation between differences in these metrics and microscopy-identified regions of live/dead cells.
Our published work using OCT built upon this prior literature to establish a quantitative, non-destructive approach to measure the 3D morphology and cell count within MCTSs breast cancer models during development10,23. Using Imaris 3D rendering image analysis software to count the number of cell-sized objects (i.e., spots) imaged within the OCT volume scans, the cell counts were non-destructively measured in MCTSs that were statistically similar to those determined via hemocytometer upon aggregate dissociation. However, due to the structural nature of OCT, cell membranes still present after cell death by necrosis may be erroneously counted as live cells. Furthermore, this characterization was extended to non-destructively track cell viability within individual aggregates subjected to a drug regimen with promising success10. Importantly, it was noted that similar cell viability was reported from our OCT-Imaris approach with what was benchmarked within these samples upon dissociation. This non-destructive and label-free cell approach enables cells to be counted within 3D constructs and dense aggregates longitudinally without sacrificing the construct/aggregate structure.
The present work reports an improved approach to directly quantify regional cell density within dense aggregates by leveraging the ability of OCT-Imaris to measure both 3D aggregate morphology and cell number. This methodological advancement provides a more detailed picture of cells&#39; spatial distribution and proliferation within the characteristic concentric layers of MCTSs models. Rather than simply calculating an overall average aggregate cell density, such local density measurements can reveal cell density gradients, such as those associated with compaction. This regional assessment is also applied to aggregates treated with a chemotherapeutic to assess regional drug response, as measured by changes in local cell density. This combination of OCT and advanced imaging analysis methods provide quantification of regional cell viability, which may be used to explore drug penetration based on which regions experience decreases in cell density. This is the first report to non-destructively quantify regional cell density and viability in response to the drug within dense cellular tissues and measure it longitudinally. Such characterization of three-dimensional cell density and spatial distribution throughout entire MCTSs may help optimize drug delivery in cancer treatment and improve the understanding of cancer model progression.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96 well plates</td>
			<td>Greiner Bio-One&#160;</td>
			<td>650970</td>
			<td>CellStar Cell-Repellent Surface, https://shop.gbo.com/en/usa/products/bioscience/cell-culture-products/cellstar-cell-repellent-surface/</td>
		</tr>
		<tr>
			<td>0.25% trypsin, 2.21 mM EDTA</td>
			<td>Corning</td>
			<td>25-053-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>AU565 breast cancer cells</td>
			<td>ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>ATCC</td>
			<td>30-2020</td>
			<td></td>
		</tr>
		<tr>
			<td>FIJI software</td>
			<td>open-source</td>
			<td>(Fiji Is Just) ImageJ v2.1/1.5.3j</td>
			<td>Downloaded from https://imagej.net/software/fiji/</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Fisher Scientific</td>
			<td>0267151B</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris image analysis software</td>
			<td>Bitplane</td>
			<td></td>
			<td>Current version 9.8</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Lonza</td>
			<td>17-605E</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354263</td>
			<td></td>
		</tr>
		<tr>
			<td>MDA-MB-231 breast cancer cells</td>
			<td>ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Zeiss</td>
			<td>Z1 AxioVision</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin streptomycin</td>
			<td>Corning</td>
			<td>30-0002CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate centrifuge</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI medium 1640</td>
			<td>Gibco</td>
			<td>11875-085</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectral Domain Optical Coherence Tomography</td>
			<td>ThorLabs</td>
			<td>TEL220C1</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 cell culture flasks</td>
			<td>Greiner Bio-One&#160;</td>
			<td>658175</td>
			<td></td>
		</tr>
		<tr>
			<td>Trastuzumab</td>
			<td></td>
			<td></td>
			<td>Remnant clinical samples of Trastuzumab were used in this study, generously gifted by the Albany Medical College Pharmacy.&#160;</td>
		</tr>
	</tbody>
</table>

non-destructive evaluation of regional cell density within tumor aggregates following drug treatment

Multicellular tumor spheroid (MCTSs) models have demonstrated increasing utility for in vitro study of cancer progression and drug discovery. These relatively simple avascular constructs mimic key aspects of in vivo tumors, such as 3D structure and pathophysiological gradients. MCTSs models can provide insights into cancer cell behavior during spheroid development and in response to drugs; however, their requisite size drastically limits the tools used for non-destructive assessment. Optical Coherence Tomography structural imaging and Imaris 3D analysis software are explored for rapid, non-destructive, and label-free measurement of regional cell density within MCTSs. This approach is utilized to assess MCTSs over a 4-day maturation period and throughout an extended 5-day treatment with Trastuzumab, a clinically relevant anti-HER2 drug. Briefly, AU565 HER2+ breast cancer MCTSs were created via liquid overlay with or without the addition of Matrigel (a basement membrane matrix) to explore aggregates of different morphologies (thicker, disk-like 2.5D aggregates or flat 2D aggregates, respectively). Cell density within the outer region, transitional region, and inner core was characterized in matured MCTSs, revealing a cell-density gradient with higher cell densities in core regions compared to outer layers. The matrix addition redistributed cell density and enhanced this gradient, decreasing outer zone density and increasing cell compaction in the cores. Cell density was quantified following drug treatment (0 h, 24 h, 5 days) within progressively deeper 100 &#181;m zones to assess potential regional differences in drug response. By the final timepoint, nearly all cell death appeared to be constrained to the outer 200 &#181;m of each aggregate, while cells deeper in the aggregate appeared largely unaffected, illustrating regional differences in the drug response, possibly due to limitations in drug penetration. The current protocol provides a unique technique to non-destructively quantify regional cell density within dense cellular tissues and measure it longitudinally.

In a prior publication, a method was established for the non-destructive measurement of global cell density within cellular aggregates using OCT10. Herein, this technique is extended to assess the regional cell density of developing cell aggregates. Figure 1 shows a schematic of this extension, where cell density can be evaluated in concentric layers of a spheroid or more locally by looking instead at small (100 &#181;m diameter) spherical plugs, denoted by the yellow...

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Non-Destructive Evaluation of Regional Cell Density Within Tumor Aggregates Following Drug Treatment

The present protocol develops an image-based technique for rapid, non-destructive, and label-free regional cell density and viability measurement within 3D tumor aggregates. Findings revealed a cell-density gradient, with higher cell densities in core regions than outer layers in developing aggregates and predominantly peripheral cell death in HER2+ aggregates treated with Trastuzumab.

Multicellular tumor spheroid (MCTSs) models have demonstrated increasing utility for in vitro study of cancer progression and drug discovery. These ...

Research

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Cancer Research

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