This work was funded by the National Institutes of Health (NIH) Award # R15CA208798 and in part by the National Science Foundation (NSF) Award # 1408007. Funding for the pulsed THz system was obtained through NSF/MRI Award # 1228958. We acknowledge the use of tissues procured by the National Disease Research Interchange (NDRI) with support from the NIH grant U42OD11158. We also acknowledge the collaboration with Oklahoma Animal Disease Diagnostic Laboratory at the Oklahoma State University for conducting the histopathology procedure on all the tissues handled in this work.

<ol>
	<li>Burford, N. M., El-Shenawee, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+terahertz+photoconductive+antenna+technology.">Review of terahertz photoconductive antenna technology.</a> Optical Engineering. 56 (1), 010901 (2017).</li><li>Sun, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+terahertz+technology+for+biomedical+applications.">Recent advances in terahertz technology for biomedical applications.</a> Quantitative Imaging in Medicine and Surgery. 7 (3), 345-355 (2017).</li><li>Wilmink, G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+investigation+of+the+biological+effects+associated+with+human+dermal+fibroblasts+exposed+to+2.52+THz+radiation.">In vitro investigation of the biological effects associated with human dermal fibroblasts exposed to 2.52 THz radiation.</a> Lasers in Surgery and Medicine. 43 (2), 152-163 (2011).</li><li>Arbab, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Terahertz+spectroscopy+for+the+assessment+of+burn+injuries+in+vivo.">Terahertz spectroscopy for the assessment of burn injuries in vivo.</a> Journal of Biomedical Optics. 18 (7), 077004 (2013).</li><li>Sy, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Terahertz+spectroscopy+of+liver+cirrhosis:+investigating+the+origin+of+contrast.">Terahertz spectroscopy of liver cirrhosis: investigating the origin of contrast.</a> Physics in Medicine and Biology. 55 (24), 7587-7596 (2010).</li><li>Yu, C., Fan, S., Sun, Y., Pickwell-Macpherson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+terahertz+imaging+for+cancer+diagnosis:+A+review+of+investigations+to+date.">The potential of terahertz imaging for cancer diagnosis: A review of investigations to date.</a> Quantitative Imaging in Medicine and Surgery. 2 (1), 33-45 (2012).</li><li>El-Shenawee, M., Vohra, N., Bowman, T., Bailey, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+detection+in+excised+breast+tumors+using+terahertz+imaging+and+spectroscopy.">Cancer detection in excised breast tumors using terahertz imaging and spectroscopy.</a> Biomedical Spectroscopy and Imaging. 8 (1-2), 1-9 (2019).</li><li>Yamaguchi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+tumor+imaging+of+rat+fresh+tissue+using+terahertz+spectroscopy.">Brain tumor imaging of rat fresh tissue using terahertz spectroscopy.</a> Scientific Reports. 6 (30124), 1-6 (2016).</li><li>Rong, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Terahertz+in-line+digital+holography+of+human+hepatocellular+carcinoma+tissue.">Terahertz in-line digital holography of human hepatocellular carcinoma tissue.</a> Scientific Reports. 5 (8445), 1-6 (2015).</li><li>Park, J. Y., Choi, H. J., Nam, G., Cho, K., Son, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+Dual-Modality+Terahertz+/+Magnetic+Resonance+Imaging+Using+Superparamagnetic+Iron+Oxide+Nanoparticles+as+a+Dual+Contrast+Agent.">In Vivo Dual-Modality Terahertz / Magnetic Resonance Imaging Using Superparamagnetic Iron Oxide Nanoparticles as a Dual Contrast Agent.</a> IEEE Transactions on Terahertz Science and Technology. 2 (1), 93-98 (2012).</li><li>Ji, Y. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feasibility+of+terahertz+reflectometry+for+discrimination+of+human+early+gastric+cancers.">Feasibility of terahertz reflectometry for discrimination of human early gastric cancers.</a> Biomedical Optics Express. 6 (4), 1413-1421 (2015).</li><li>Bowman, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Phantom+Study+of+Terahertz+Spectroscopy+and+Imaging+of+Micro-+and+Nano-diamonds+and+Nano-onions+as+Contrast+Agents+for+Breast+Cancer.">A Phantom Study of Terahertz Spectroscopy and Imaging of Micro- and Nano-diamonds and Nano-onions as Contrast Agents for Breast Cancer.</a> Biomedical Physics and Engineering Express. 3 (5), 055001 (2017).</li><li>Chavez, T., Bowman, T., Wu, J., Bailey, K., El-Shenawee, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+Terahertz+Imaging+for+Excised+Breast+Cancer+Tumors+with+Image+Morphing.">Assessment of Terahertz Imaging for Excised Breast Cancer Tumors with Image Morphing.</a> Journal of Infrared, Millimeter, and Terahertz Waves. 39 (12), 1283-1302 (2018).</li><li>Bowman, T. C., El-Shenawee, M., Campbell, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Terahertz+Imaging+of+Excised+Breast+Tumor+Tissue+on+Paraffin+Sections.">Terahertz Imaging of Excised Breast Tumor Tissue on Paraffin Sections.</a> IEEE Transactions on Antennas and Propagation. 63 (5), 2088-2097 (2015).</li><li>Bowman, T., El-Shenawee, M., Campbell, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Terahertz+transmission+vs+reflection+imaging+and+model-based+characterization+for+excised+breast+carcinomas.">Terahertz transmission vs reflection imaging and model-based characterization for excised breast carcinomas.</a> Biomedical Optics Express. 7 (9), 3756-3783 (2016).</li><li>Bowman, T., Wu, Y., Gauch, J., Campbell, L. K., El-Shenawee, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Terahertz+Imaging+of+Three-Dimensional+Dehydrated+Breast+Cancer+Tumors.">Terahertz Imaging of Three-Dimensional Dehydrated Breast Cancer Tumors.</a> Journal of Infrared, Millimeter, and Terahertz Waves. 38 (6), 766-786 (2017).</li><li>Bowman, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulsed+terahertz+imaging+of+breast+cancer+in+freshly+excised+murine+tumors.">Pulsed terahertz imaging of breast cancer in freshly excised murine tumors.</a> Journal of Biomedical Optics. 23 (2), 026004 (2018).</li><li>Bowman, T., Vohra, N., Bailey, K., El-Shenawee, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Terahertz+tomographic+imaging+of+freshly+excised+human+breast+tissues.">Terahertz tomographic imaging of freshly excised human breast tissues.</a> Journal of Medical Imaging. 6 (2), 023501 (2019).</li><li>Vohra, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulsed+Terahertz+Reflection+Imaging+of+Tumors+in+a+Spontaneous+Model+of+Breast+Cancer.">Pulsed Terahertz Reflection Imaging of Tumors in a Spontaneous Model of Breast Cancer.</a> Biomedical Physics and Engineering Express. 4 (6), 065025 (2018).</li><li>Jacobs, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+margins:+the+challenge+continues+for+breast+surgeons.">Positive margins: the challenge continues for breast surgeons.</a> Annals of Surgical Oncology. 15 (5), 1271-1272 (2008).</li><li>Moran, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Society+of+Surgical+Oncology--American+Society+for+Radiation+Oncology+Consensus+Guideline+on+Margins+for+Breast-Conserving+Surgery+With+Whole-Breast+Irradiation+in+Stages+I+and+II+Invasive+Breast+Cancer.">Society of Surgical Oncology--American Society for Radiation Oncology Consensus Guideline on Margins for Breast-Conserving Surgery With Whole-Breast Irradiation in Stages I and II Invasive Breast Cancer.</a> International Journal of Radiation Oncology. 88 (3), 553-564 (2014).</li><li>Fitzgerald, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Terahertz+Pulsed+Imaging+of+human+breast+tumors.">Terahertz Pulsed Imaging of human breast tumors.</a> Radiology. 239 (2), 533-540 (2006).</li><li>Ashworth, P. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Terahertz+pulsed+spectroscopy+of+freshly+excised+human+breast+cancer.">Terahertz pulsed spectroscopy of freshly excised human breast cancer.</a> Optics Express. 17 (15), 12444-12454 (2009).</li><li>Doradla, P., Alavi, K., Joseph, C., Giles, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+colon+cancer+by+continuous-wave+terahertz+polarization+imaging+technique.">Detection of colon cancer by continuous-wave terahertz polarization imaging technique.</a> Journal of Biomedical Optics. 18 (9), 090504 (2013).</li><li>Reid, C. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Terahertz+pulsed+imaging+of+freshly+excised+human+colonic+tissues.">Terahertz pulsed imaging of freshly excised human colonic tissues.</a> Physics in Medicine and Biology. 56 (1), 4333-4353 (2011).</li><li>. Teraview.com Available from: <a target="_blank" href="https://teraview.com">https://teraview.com</a> (2019)</li><li>Orosco, R. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+Surgical+Margins+in+the+10+Most+Common+Solid+Cancers.">Positive Surgical Margins in the 10 Most Common Solid Cancers.</a> Scientific Reports. 8 (1), 1-9 (2018).</li><li>Bowman, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistical+signal+processing+for+quantitative+assessment+of+pulsed+terahertz+imaging+of+human+breast+tumors.">Statistical signal processing for quantitative assessment of pulsed terahertz imaging of human breast tumors.</a> 2017 42nd International Conference on Infrared, Millimeter, and Terahertz Waves (IRMMW-THz). , 1-2 (2017).</li><li>Gavdush, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Terahertz+spectroscopy+of+gelatin-embedded+human+brain+gliomas+of+different+grades:+a+road+toward+intraoperative+THz+diagnosis.">Terahertz spectroscopy of gelatin-embedded human brain gliomas of different grades: a road toward intraoperative THz diagnosis.</a> Journal of Biomedical Optics. 24 (2), 027001 (2019).</li></ol>

The authors declare that they have no conflict of interest.

Effective THz reflection imaging of fresh tissue is primarily dependent on two critical aspects: 1) the proper consideration of tissue handling (sections 2 and 4.15); and 2) the stage setup (primarily section 4.11). Insufficient drying of the tissue can result in increased reflection and inability to visualize regions due to high reflections of DMEM and other fluids. Meanwhile, poor tissue contact with the imaging window creates rings or spots of low reflection in the THz reflection image that obscure the results. Extra ...

Terahertz (THz) imaging and spectroscopy has been a rapidly growing area of research in the past decade. The continued development of more efficient and consistent THz emitters in the range of 0.1&#8211;4 THz has made their applications grow significantly1. One area where THz has shown promise and significant growth is the biomedical field2. THz radiation has been shown to be nonionizing and biologically safe at the power levels generally used to analyze fixed tissues3. As a result, THz imaging and spectroscopy has been used to classify and differentiate various tissue features such as water content to indicate burn damage and healing4, liver cirrhosis5, and cancer in excised tissues6,7. Cancer assessment in particular covers a broad range of potential clinical and surgical applications, and has been investigated for cancers of the brain8, liver9, ovaries10, gastrointestinal tract11, and breast7,12,13,14,15,16,17,18,19.
THz applications for breast cancer are primarily focused on supporting breast conserving surgery, or lumpectomy, via margin assessment. The objective of a lumpectomy is to remove the tumor and a small layer of surrounding healthy tissue, in contrast to full mastectomy, which removes the entire breast. The surgical margin of the excised tissue is then assessed via pathology once the sample has been fixed in formalin, sectioned, embedded in paraffin, and mounted in 4 &#181;m&#8211;5 &#181;m slices on microscope slides. This process can be time-consuming and requires a secondary surgical procedure at a later time if a positive margin is observed20. Current guidelines by the American Society of Radiation Oncology define this positive margin as having cancer cells contacting the surface-level margin ink21. THz imaging for high-absorption hydrated tissue is primarily limited to surface imaging with some varying penetration based on tissue type, which is sufficient for meeting the surgical needs of rapid margin assessment. A quick analysis of margin conditions during the surgical setting would greatly decrease surgical costs and follow-up procedure rate. To date, THz has proven effective in differentiating between cancer and healthy tissue in formalin-fixed, paraffin-embedded (FFPE) tissues, but additional investigation is needed to provide reliable detection of cancer in freshly excised tissues7.
This protocol details the steps for performing THz imaging and spectroscopy on freshly excised human tissue samples obtained from a biobank. THz applications built on freshly excised human breast cancer tissues have seldom been used in published research7,18,22,23, especially by research groups not integrated with a hospital. The use of freshly excised tissues is likewise rare for other cancer applications, with most non-breast human cancer examples being reported for colonic cancer24,25. One reason for this is that FFPE tissue blocks are far easier to access and handle than freshly excised tissue unless the THz system being used for the study is part of the surgical workflow. Similarly, most commercial laboratory THz systems are not prepared to handle fresh tissue, and those that do are still in the stages of using cell line growth or have only started to look at excised tissue from animal models. To apply THz to an intraoperative setting requires that imaging and characterization steps be developed for fresh tissue in advance so that the analysis does not interfere with the ability to perform standard pathology. For applications that are not inherently meant to be intraoperative, the characterization of fresh tissue is still a challenging step that must be addressed to work towards in vivo applications and differentiation.
The objective of this work is to provide a guideline for THz application for freshly excised tissue using a commercial THz system. The protocol was developed on a THz imaging and spectroscopy system26 for murine breast cancer tumors13,17,19 and was extended to human surgical tissue obtained from biobanks7,18. While the protocol was generated for breast cancer, the same concepts can be applied to similar THz imaging systems and other types of solid-tumor cancers that are treated with surgery where success depends on margin assessment27. Due to a fairly small amount of published THz results on freshly excised tissues, this is the first work to the authors&#8217; knowledge to focus on the protocol of fresh tissue handling for THz imaging and characterization.

<table><tbody><tr><td>70% isopropyl alcohol</td><td>VWR</td><td>89108-162</td><td>Contains 70% USP grade isopropanol and 30% USP grade deionized water</td></tr><tr><td>Alconox powder detergent</td><td>VWR</td><td>21835-032</td><td>Concentrated detergent to remove organic contaminants from glass, metal, stainless steel, porcelain, ceramic, plastic, rubber, and fiberglass</td></tr><tr><td>Bio Hazard Bags</td><td>Fisher Scientific</td><td>19-033-712</td><td>Justrite FM-Approved Biohazard Waste Container Replacement Bags</td></tr><tr><td>Cardboard holder</td><td>N/A</td><td>N/A</td><td>Scrap cardboard to keep tissue imaging face intact when immersed in formalin</td></tr><tr><td>Centrifuge Tubes</td><td>VWR</td><td>10026-078</td><td>Centrifuge Tubes with Flat Caps, Conical-Bottom, Polypropylene, Sterile, Standard Line</td></tr><tr><td>Cotton Swabs</td><td>Walmart</td><td>551398298</td><td>Q-tips Original Cotton Swabs used to dye the tissue</td></tr><tr><td>Ethyl Alcohol</td><td>VWR</td><td>71002-426</td><td>KOPTECH Pure (undenatured) anhydrous (200 proof/100%) ethyl alcohol</td></tr><tr><td>Eye protection goggles</td><td>VWR</td><td>89130-918</td><td>Kimberly-clark professional safety glasses</td></tr><tr><td>Face Mask</td><td>VWR</td><td>95041-774</td><td>DUKAL Corporation surgical masks</td></tr><tr><td>Filter paper</td><td>Sigma Aldrich</td><td>Z240087</td><td>Whatman grade 1 cellulose filters</td></tr><tr><td>Formalin solution</td><td>Sigma Aldrich</td><td>HT501128-4L</td><td>10% neutral buffered formalin</td></tr><tr><td>Human freshly excised tumors (Infilterating Ductal Carcinoma (IDC))</td><td>National Disease Research Interchange (NDRI biobank</td><td>N/A</td><td>A protocol is signed with the NDRI for the type of tumors required</td></tr><tr><td>IRADECON Bleach solution</td><td>VWR</td><td>89234-816</td><td>Pre-diluted Sodium Hypochlorite Bleach solution</td></tr><tr><td>KIMTECH SCIENCE wipes</td><td>VWR</td><td>21905-026</td><td>Kimberly-clark professional Kim wipes</td></tr><tr><td>Laboratory Coat</td><td>VWR</td><td>10141-342</td><td>This catalog number is for medium size coat</td></tr><tr><td>Laboratory tweezers/Forceps</td><td>VWR</td><td>82027-388</td><td>Any laboratory tweezers can be used as long as it does not damage the tissue</td></tr><tr><td>Liquid sample holder (two quartz windows with a 0.1 mm teflon spacer)</td><td>TeraView, Ltd</td><td>N/A</td><td>1&quot; diameter, and 0.1452&quot; thick quartz windows</td></tr><tr><td>Nitrile hand gloves</td><td>VWR</td><td>82026-426</td><td>This catalog number is for medium size gloves</td></tr><tr><td>Nitrogen cylinder</td><td>Airgas</td><td>NI UHP300</td><td>NITROGEN UHP GR 5.0 SIZE 300</td></tr><tr><td>Paper towel</td><td>VWR</td><td>14222-321</td><td>11&quot; x 8.78&quot; Sheets, 1 Ply</td></tr><tr><td>Parafilm</td><td>VWR</td><td>52858-076</td><td>Flexible thermoplastic. Rolled, waterproof sheet interwound with paper to prevent self-adhesion.</td></tr><tr><td>Petri Dish</td><td>VWR</td><td>470210-568</td><td>VWR Petri Dish, Slippable, Mono Plate (undivided bottom)</td></tr><tr><td>Polystyrene Plate</td><td>Home Depot</td><td>1S11143A</td><td>~ 10 cm x 10 cm square piece cut from a 11&quot; x 14&quot; x 0.05&quot; Non-glare styrene sheet</td></tr><tr><td>ScanAcquire Software</td><td>TeraView, Ltd</td><td>N/A</td><td>System Software for THz reflection imaging measurements</td></tr><tr><td>Stainless steel low-profile blade (#4689)</td><td>VWR</td><td>25608-964</td><td>Tissue-Tek Accu-Edge Disposable Microtome Blades</td></tr><tr><td>Stainless steel metal tray</td><td>Quick Medical</td><td>10F</td><td>Polar Ware Stainless Steel Medical Instrument Trays</td></tr><tr><td>Tissue Marking Dyes</td><td>Ted Pella, Inc</td><td>Yellow Dye #27213-1&lt;br/&gt; Red Dye #27213-2&lt;br/&gt; Blue Dye #27213-4</td><td>Used to orient excised tissue samples&lt;br/&gt; sent to the histopathology laboratory</td></tr><tr><td>TPS Spectra 3000</td><td>TeraView, Ltd</td><td>N/A</td><td>THz imaging and spectroscopy system</td></tr><tr><td>TPS Spectra Software</td><td>TeraView, Ltd</td><td>N/A</td><td>System Software for THz transmission spectroscopy measurements</td></tr></tbody></table>

terahertz imaging and characterization protocol for freshly excised breast cancer tumors

This manuscript presents a protocol to handle, characterize, and image freshly excised human breast tumors using pulsed terahertz imaging and spectroscopy techniques. The protocol involves terahertz transmission mode at normal incidence and terahertz reflection mode at an oblique angle of 30&#176;. The collected experimental data represent time domain pulses of the electric field. The terahertz electric field signal transmitted through a fixed point on the excised tissue is processed, through an analytical model, to extract the refractive index and absorption coefficient of the tissue. Utilizing a stepper motor scanner, the terahertz emitted pulse is reflected from each pixel on the tumor providing a planar image of different tissue regions. The image can be presented in time or frequency domain. Furthermore, the extracted data of the refractive index and absorption coefficient at each pixel are utilized to provide a tomographic terahertz image of the tumor. The protocol demonstrates clear differentiation between cancerous and healthy tissues. On the other hand, not adhering to the protocol can result in noisy or inaccurate images due to the presence of air bubbles and fluid remains on the tumor surface. The protocol provides a method for surgical margins assessment of breast tumors.

The THz imaging results18 obtained following the abovementioned protocol of human breast cancer tumor specimen #ND14139 received from the biobank are presented in Figure 9. According to the pathology report, the #ND14139 tumor was a I/II grade infiltrating ductal carcinoma (IDC) obtained from a 49-year-old woman via a left breast lumpectomy surgery procedure. The photograph of the tumor is shown in Figure 9A, the pathology image in <stron...

Watch this Scientific Journal Video about Terahertz Imaging and Characterization Protocol for Freshly Excised Breast Cancer Tumors at JoVE.com

Terahertz Imaging and Characterization Protocol for Freshly Excised Breast Cancer Tumors

Freshly excised human breast cancer tumors are characterized with terahertz spectroscopy and imaging following fresh tissue handling protocols. Tissue positioning is taken into consideration to enable effective characterization while providing analysis in a timely manner for future intraoperative applications.

This manuscript presents a protocol to handle, characterize, and image freshly excised human breast tumors using pulsed terahertz imaging and spectroscopy ...

terahertz-imaging-characterization-protocol-for-freshly-excised

Research

JoVE Journal

Engineering

10.8K Views.  University of Arkansas. Freshly excised human breast cancer tumors are characterized with terahertz spectroscopy and imaging following fresh tissue handling protocols. Tissue positioning is taken into consideration to enable effective characterization while providing analysis in a timely manner for future intraoperative applications.

Video: Terahertz Imaging and Characterization Protocol for Freshly Excised Breast Cancer Tumors

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Medicine

Using Computer-based Image Analysis to Improve Quantification of Lung Metastasis in the 4T1 Breast Cancer Model

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. The leading cause of breast cancer related deaths is the metastatic burden. Therefore, preclinical models for breast cancer need to analyze metastatic burden to be clinically relevant. The 4T1 breast cancer model provides a spontaneously-metastasizing, quantifiable mouse model for stage IV human breast cancer. However, most 4T1 protocols quantify the metastatic burden by manually counting stained colonies on tissue culture plates. While this is sufficient for tissues with lower metastatic burden, human error in manual counting causes inconsistent and variable results when plates are confluent and difficult to count. This method offers a computer-based solution to human counting error. Here, we evaluate the protocol using the lung, a highly metastatic tissue in the 4T1 model. Images of methylene blue-stained plates are acquired and uploaded for analysis in Fiji-ImageJ. Fiji-ImageJ then determines the percentage of the selected area of the image that is blue, representing the percentage of the plate with metastatic burden. This computer-based approach offers more consistent and expeditious results than manual counting or histopathological evaluation for highly metastatic tissues. The consistency of Fiji-ImageJ results depends on the quality of the image. Slight variations in results between images can occur, thus it is recommended that multiple images are taken and results averaged. Despite its minimal limitations, this method is an improvement to quantifying metastatic burden in the lung by offering consistent and rapid results.

We describe a more consistent and expeditious method to quantify lung metastasis in the 4T1 breast cancer model by using Fiji-ImageJ.

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. ...

Cancer Research

Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 months. It is critical to have suitable animal models to mimic the hemodynamic spread of the metastatic cells in the clinical scenario. Here, we are introducing the use of small animal ultrasound imaging to guide an accurate injection of brain tropical breast cancer cells into the left ventricle of athymic nude mice. Longitudinal MRI is used to assessing intracranial initiation and growth of brain metastases. Ultrasound-guided intracardiac injection ensures not only an accurate injection and hereby a higher successful rate but also significantly decreased mortality rate, as compared to our previous manual procedure. In vivo high resolution MRI allows the visualization of hyperintense multifocal lesions, as small as 310 &#181;m in diameter on T2-weighted images at 3 weeks post injection. Follow-up MRI reveals intracranial tumor growth and increased number of metastases that distribute throughout the whole brain.

A breast cancer brain metastasis mouse model is established with ultrasound imaging-guided intracardiac injection of MDA-MB231/Br-GFP cells. Development of multifocal intracranial metastases has been monitored longitudinally using high-resolution 9.4 T MRI.

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 ...

Tissue-simulating Phantoms for Assessing Potential Near-infrared Fluorescence Imaging Applications in Breast Cancer Surgery

Inaccuracies in intraoperative tumor localization and evaluation of surgical margin status result in suboptimal outcome of breast-conserving surgery (BCS). Optical imaging, in particular near-infrared fluorescence (NIRF) imaging, might reduce the frequency of positive surgical margins following BCS by providing the surgeon with a tool for pre- and intraoperative tumor localization in real-time. In the current study, the potential of NIRF-guided BCS is evaluated using tissue-simulating breast phantoms for reasons of standardization and training purposes.
Breast phantoms with optical characteristics comparable to those of normal breast tissue were used to simulate breast conserving surgery. Tumor-simulating inclusions containing the fluorescent dye indocyanine green (ICG) were incorporated in the phantoms at predefined locations and imaged for pre- and intraoperative tumor localization, real-time NIRF-guided tumor resection, NIRF-guided evaluation on the extent of surgery, and postoperative assessment of surgical margins. A customized NIRF camera was used as a clinical prototype for imaging purposes.
Breast phantoms containing tumor-simulating inclusions offer a simple, inexpensive, and versatile tool to simulate and evaluate intraoperative tumor imaging. The gelatinous phantoms have elastic properties similar to human tissue and can be cut using conventional surgical instruments. Moreover, the phantoms contain hemoglobin and intralipid for mimicking absorption and scattering of photons, respectively, creating uniform optical properties similar to human breast tissue. The main drawback of NIRF imaging is the limited penetration depth of photons when propagating through tissue, which hinders (noninvasive) imaging of deep-seated tumors with epi-illumination strategies.

Near-infrared fluorescence (NIRF) imaging may improve therapeutic outcome of breast cancer surgery by enabling intraoperative tumor localization and evaluation of surgical margin status. Using tissue-simulating breast phantoms containing fluorescent tumor-simulating inclusions, potential clinical applications of NIRF imaging in breast cancer patients can be assessed for standardization and training purposes.

Inaccuracies in intraoperative tumor localization and evaluation of surgical margin status result in suboptimal outcome of breast-conserving surgery ...

Tracking the Mammary Architectural Features and Detecting Breast Cancer with Magnetic Resonance Diffusion Tensor Imaging

Breast cancer is the most common cause of cancer among women worldwide. Early detection of breast cancer has a critical role in improving the quality of life and survival of breast cancer patients. In this paper a new approach for the detection of breast cancer is described, based on tracking the mammary architectural elements using diffusion tensor imaging (DTI). 
The paper focuses on the scanning protocols and image processing algorithms and software that were designed to fit the diffusion properties of the mammary fibroglandular tissue and its changes during malignant transformation. The final output yields pixel by pixel vector maps that track the architecture of the entire mammary ductal glandular trees and parametric maps of the diffusion tensor coefficients and anisotropy indices. 
The efficiency of the method to detect breast cancer was tested by scanning women volunteers including 68 patients with breast cancer confirmed by histopathology findings. Regions with cancer cells exhibited a marked reduction in the diffusion coefficients and in the maximal anisotropy index as compared to the normal breast tissue, providing an intrinsic contrast for delineating the boundaries of malignant growth. Overall, the sensitivity of the DTI parameters to detect breast cancer was found to be high, particularly in dense breasts, and comparable to the current standard breast MRI method that requires injection of a contrast agent. Thus, this method offers a completely non-invasive, safe and sensitive tool for breast cancer detection.

We describe how to obtain parametric and vector maps of the diffusion tensor of the breast using magnetic resonance imaging. The protocol and final output following imaging processing are tailored for tracking breast architectural features and detecting breast malignancy.

Breast cancer is the most common cause of cancer among women worldwide. Early detection of breast cancer has a critical role in improving the quality of ...

Radiation Response in Patient-Derived Tumor Organoids Using a Clonogenic Assay

Live Imaging to Quantify Cellular Radiosensitivity in Patient-Derived Tumor Organoids

Radiation therapy (RT) is one of the mainstays of modern clinical cancer management. However, not all cancer types are equally sensitive to irradiation, often (but not always) because of differences in the ability of malignant cells to repair oxidative DNA damage as elicited by ionizing rays. Clonogenic assays have been employed for decades to assess the sensitivity of cultured cancer cells to ionizing irradiation, largely because irradiated cancer cells often die in a delayed manner that is difficult to quantify with short-term flow cytometry- or microscopy-assisted techniques. Unfortunately, clonogenic assays cannot be employed as such for more complex tumor models, such as patient-derived tumor organoids (PDTOs). Indeed, irradiating established PDTOs may not necessarily abrogate their growth as multicellular units, unless their stem-like compartment is completely eradicated. Moreover, irradiating PDTO-derived single-cell suspensions may not properly recapitulate the sensitivity of malignant cells to RT in the context of established PDTOs. Here, we detail an adaptation of conventional clonogenic assays that involves exposure of established PDTOs to ionizing radiation, followed by single-cell dissociation, replating in suitable culture conditions and live imaging. Non-irradiated (control) PDTO-derived stem-like cells reform growing PDTOs with a PDTO-specific efficiency, which is negatively influenced by irradiation in a dose-dependent manner. In these conditions, PDTO-forming efficiency and growth rate can be quantified as a measure of radiosensitivity on time-lapse images collected until control PDTOs achieve a predefined space occupancy.

Author Spotlight: Radiotherapy and Clonogenic Assays for Advancing Cancer Research and Personalized Medicine

Here, we detail a straightforward live imaging approach for quantifying the sensitivity of patient-derived tumor organoids to ionizing radiation.

Radiation therapy (RT) is one of the mainstays of modern clinical cancer management. However, not all cancer types are equally sensitive to irradiation, ...

High-Throughput Dissociation and Orthotopic Implantation of Breast Cancer Patient-Derived Xenografts

Patient-derived xenografts (PDXs) provide a clinically relevant method for recapitulating tumor-involved cell types and the tumor microenvironment, which is essential for advancing knowledge of breast cancer (BC). Additionally, PDX models enable the study of BC systemic effects, which is not possible using in vitro models. Traditional methods for implanting BC xenografts typically involve anesthesia and sterile surgical procedures, which are time-consuming, invasive, and limit the scalability of PDX models in BC research. This protocol describes a simple and scalable method for the orthotopic implantation of BC PDXs in mice. The immunodeficient mouse strain NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) was used for PDX engraftment. Human BC samples obtained from IRB-consented patients were mechanically and enzymatically dissociated, then resuspended in a solution of basement membrane extract (BME) and RPMI 1640. Animals were restrained by scruffing, and depilatory cream was applied to remove hair from the fat pads at the fourth inguinal nipple, followed by injection. Approximately 2 million cells in a 100 &#181;L suspension were bilaterally injected orthotopically into the mammary fat pads using a 26 G needle. Notably, no anesthetic was required, and the total procedure time was under 5 min, from cell preparation to injection. After a growth period of several months, tumors were excised and processed for authentication. Validation included receptor status assessment using immunohistochemistry with specific antibodies for traditional BC receptors (i.e., ER, PR, HER2). Tumor morphology was confirmed with hematoxylin and eosin (H&amp;E) staining, which was interpreted by a pathologist. Genetic similarity to the patient sample was verified through bulk RNA sequencing and short tandem repeat (STR) analysis. This approach to PDX engraftment and validation supports the rigorous development of models and high-throughput tumor implantation, enabling well-powered studies across various BC subtypes.

This protocol describes an efficient, non-surgical method for the orthotopic implantation of breast cancer patient-derived xenografts in mice. The technique involves enzymatic tumor dissociation followed by direct injection into the mammary fat pads, enabling high-throughput implantation. Comprehensive validation ensures model fidelity, facilitating rigorous studies across various breast cancer subtypes.

Patient-derived xenografts (PDXs) provide a clinically relevant method for recapitulating tumor-involved cell types and the tumor microenvironment, which ...

Three-Dimensional Imaging of Tumor-Bearing Tissue Using the Iterative Bleaching Extends Multiplexity Approach

The spatial heterogeneity of the tumor microenvironment (TME) is a critical determinant of therapeutic response, particularly for immune-oncology agents, where success depends on the distribution of specific immune cell subpopulations. Over the past decade, multiple sophisticated technologies have been introduced to achieve detailed resolution of the TME using two-dimensional sections from either formalin-fixed, paraffin-embedded (FFPE) or fixed-frozen tissues. While these thin sections are easier to procure and analyze, they lack the three-dimensional architecture needed to reliably and comprehensively characterize a tumor. To address this limitation, a tissue mounting and imaging technique was developed to enable the three-dimensional analysis of tumor lesions in their native in vivo state. This protocol outlines the procurement of human tumor tissue, the mounting of samples on custom-printed platforms, and staining procedures for post-fixation samples. The multiplexed immunofluorescence technique, IBEX (Iterative Bleaching Extends Multiplexity), was adapted to characterize the three-dimensional TME with up to 15 markers for tumor, immune, and stromal cells using commercially available antibodies. Imaging depths of up to 100 &#181;m were achieved using an inverted white-light laser confocal microscope with a custom-printed imaging adaptor and commercial glass-bottom dishes to ensure optimal tissue orientation. This protocol highlights the potential of the IBEX method to expand multiplexed immunofluorescence studies, providing a more comprehensive understanding of TME composition.

This article describes a protocol for multiplex immunofluorescence optimized to characterize the three-dimensional architecture of peritoneal metastases.

The spatial heterogeneity of the tumor microenvironment (TME) is a critical determinant of therapeutic response, particularly for immune-oncology agents, ...

Plasmonic Photothermal Cancer Therapy: Nanoparticle-embedded Tumor-tissue-mimicking Phantoms for Visualizing Photothermal Temperature Distribution

Plasmonic photothermal therapy (PPTT), an emerging cancer treatment, involves delivering nanoparticles (NPs) to a tumor, followed by near-infrared (NIR) irradiation to generate localized heat that destroys cancer cells. Before administering PPTT, the therapeutic parameters -- NP concentration, irradiation intensity, and duration -- need to be estimated. For this, numerical simulations are performed. However, to ensure robust computation, these simulations must be validated through photothermal experiments on tumor-tissue-mimicking phantoms replicating the optical properties of tumor tissue. For PPTT, therapeutic parameters are governed by the scattering and absorption of incident radiation by the tissue and NPs. Therefore, validation experiments can be conducted on phantoms mimicking the reduced scattering coefficient (&#181;s&#39;) and absorption coefficient (&#181;a) of the target tumor/tissue.
Specifically, this protocol provides instructions for preparing phantoms mimicking &#181;s&#39; and &#181;a of breast tumor injected with gold nanorods, surrounded by normal breast tissue. The protocol also details NIR irradiation, temperature monitoring, and validation of numerical results by comparing spatiotemporal temperatures with those measured using thermocouples. The protocols presented in this study facilitated the preparation of hydrogel-based cylindrical breast tumor-tissue phantoms with dimensions (&#981;40 x 12 mm) and a central tumor region (&#981;20 x 6 mm), comprising 1% agarose as the base matrix and intralipid as the scattering constituent and tumor region embedded with gold nanorods at 25 &#181;g/mL concentration. Representative results from a case study illustrate the application of fabricated phantoms for validating numerical simulations for PPTT. The study concludes that the demonstrated protocols are valuable for conducting photothermal experiments aimed at optimizing and planning therapeutic parameters prior to in vivo experiments and validating numerical simulations for PPTT.

This article presents the protocol for preparing tumor-tissue phantoms that replicate optical properties for plasmonic photothermal therapy. It details phantom preparation, photothermal evaluations, and validation of the developed numerical model based on photothermal temperature measurements for assessing therapeutic parameters, offering an ethical, cost-effective alternative to in vivo studies for preliminary testing.

Plasmonic photothermal therapy (PPTT), an emerging cancer treatment, involves delivering nanoparticles (NPs) to a tumor, followed by near-infrared (NIR) ...

Building Up a High-throughput Screening Platform to Assess the Heterogeneity of HER2 Gene Amplification in Breast Cancers

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast cancers. However, a minority of cases displays a heterogeneous distribution of HER2-positive cells, which generates major clinical challenges. To date, no reliable and standardized protocols for the characterization and quantification of HER2 heterogeneous gene amplification in large cohorts have been proposed. Here, we present a high-throughput methodology to simultaneously assess the HER2 status across different topographic areas of multiple breast cancers. In particular, we illustrate the laboratory procedure to construct enhanced tissue microarrays (TMAs) incorporating a targeted mapping of the tumors. All TMA parameters have been specifically optimized for the silver in situ hybridization (SISH) of formalin-fixed paraffin-embedded (FFPE) breast tissues. Immunohistochemical analysis of the prognostic and predictive biomarkers (i.e., ER, PR, Ki67, and HER2) should be performed using automated procedures. A customized SISH protocol has been implemented to allow a high-quality molecular analysis across multiple tissues that underwent different fixation, processing, and storage procedures. In this study, we provide a proof-of-principle that specific DNA sequences could be localized simultaneously in distinct topographic areas of multiple and heterogeneously processed breast cancers using an efficient and cost-effective method.

Heterogeneous distribution of HER2-positive cells can be observed in a subset of breast cancers and generates clinical dilemmas. Here, we introduce a reliable and cost-effective protocol to define, quantify, and compare HER2 intra-tumor genetic heterogeneity in a large series of heterogeneously processed breast cancers.

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast ...