INQUIRY VOLUME 19 Terahertz Imaging Platform to Characterize the Growth of In-Vitro Breast Tumors

Transcription

1 INQUIRY VOLUME 9 Terhertz Imging Pltform to Chrcterize the Growth of In-Vitro Brest Tumors By: Scrlett-Mrie Acklin Deprtment of Biologicl Sciences Fculty Mentor: Dr. Mgd El-Shenwee Deprtment of Electricl Engineering Astrct This study imed t evluting the idel plting method nd density for imging with the terhertz (THz) spectrometer. In this study, different methods were used to grow in-vitro tumors using the T cell line. Here, ttempts to grow rest tumors in-vitro were conducted. Results were produced in two environments, flt-ottomed pltes nd round-ottomed multiwell pltes. The second method llowed for fster clumping nd incresed cell ggregtion, producing tumors up to 7mm. Terhertz spectroscopy produced imges tht correlted well to photomicrogrphs tken of the in-vitro tumors. This methodology shows gret promise for providing relile, prmeter-controlled source of in-vitro rest tumors for reserch needs on rest tumor mrgins. Introduction Becuse rest cncer is the most common mlignnt disese in women (Weigelt et l., 25), reserch to develop more effective tretments is extremely importnt. For the yer 25, the Americn Cncer Society predicts 23,9 new cses of rest cncer will e dignosed nd,73 deths will occur (Americn Cncer Society, 25). This sttistic demonstrtes n increse, rther thn decrese, in new cses of rest cncer from the yer 2. Routine screenings for rest cncer hve ecome common prctice in the United Sttes since the 96s, nd x-ry mmmogrphy hs een the primry method used (Hssn & El-Shenwee, 2). The use of x-ry mmmogrphy comes with disdvntges; s result, lterntives re eing sought. For exmple, x-rys pose helth risk due to ionizing cpilities which re known to produce free rdicls, rek nd form chemicl onds, nd dmge vitl molecules such s deoxyrionucleic cid, rionucleic cid, nd proteins (Office of Environmentl Helth nd Sfety, 25). This type of cell dmge cn led to n increse in cncer development (Alerts et l., 22), therey compounding the prolem. Additionlly, the inccurcy of x-ry mmmogrphy is high, rnging from % to 3% (Huynh et l., 998). Mgnetic resonnce imging (MRI) exists s n lterntive to x-rys, ut only for high-risk ptients due to its expensive operting costs nd its limited ility to distinguish etween norml cells nd cncerous cells, oftentimes leding to overdignosis (Hssn & El-Shenwee, 2). Removing the cncer efore metstsis is lso crucil, ecuse metstses in the lymph nodes nd other vitl orgns such s the lungs, liver, rin, nd ones cuse most of the complictions linked to rest cncer, including deth (Fntozzi & Christofori, 26). During lumpectomy to remove smll tumors, region surrounding the tumor, clled the mrgin (Fig. ) (Brestcncer.org, 25) nd (Bowmn et l., 25), is removed. A pthologist then exmines the mrgin for cncerous cells. To mintin the est cosmetic ppernce, it is importnt to differentite etween cncer, helthy firous nd glndulr tissue immeditely surrounding the tumor, nd helthy ftty rest tissue (Bowmn et l., 25). A positive mrgin, requiring second surgery, demonstrtes tht cncer hs extended cross the edge of the excised tumor. The two other clssifictions do not require secondry surgery. The first of these is close mrgin denoting cncer within 2mm of the tumor s edge nd the second is negtive mrgin, where no cncer is detected within 2mm of the order. Positive mrgins not only cuse cosmetic nd emotionl dmge for the ptient, ut they re lso very expensive due to the time nd resources needed to crry out second surgery. Reserch studies indicte tht positive mrgins re detected 2-% of the time (Pleijhuis et l., 29). A new medicl imging modlity, ville t the University of Arknss, uses THz wves to develop high-resolution imges nd chrcteriztion of humn rest tumors fixed in formlin nd emedded in prffin. THz wves mesure from one mm to one µm, etween infrred light nd microwves. Their 53

2 for studying humn rest cncer ecuse metstsis occurs rndomly from the initil tumor, the cells re trnsplntle so they cn lso e grown in in vivo environments, nd its spred to the lymph nodes nd other orgns is similr to the metstsis in humn rest cncer (Pulski & Ostrnd-Rosenerg, 2). Methods nd Mterils % FBS DMEM medi nd T cell culture Figure. Animtions of positive nd negtive mrgins (Brest Cncer Orgniztion). wvelengths hve the medicl dvntge of possessing energy levels lower thn the levels in x-rys tht ionize cells (Burford et l., 2). THz wvelengths hve nother dvntge in tht they re shorter thn millimeter wves nd therefore, generte higher imge resolution while still penetrting the smple (Jepsen et l., 996). Finlly, THz wves produce limited levels of scttering in iologicl mterils due to their longer wvelengths, insted of the strong scttering chrcteristic of nerinfrred nd visile wvelengths. Photons re scttered more strongly y iologicl mterils ecuse their size mtches the incident wvelength (Wilmink & Grundt, 2). For nlysis, THz pulse is emitted nd strikes the smple, then receiving ntenn mesures the intensity of the reflection off of the smple (Burford et l., 2). To further exmine this new imging system in lortory environment, ttempts to grow rest tumors in-vitro were conducted. Current methods drip cells from n upper surfce (Sto et l., 977) or use multilevel chmers to pss the cells from n upper chmer, through smll pore, nd into ottom chmer where they re suspended from the roof (Dimri et l., 27). The mterils used in these methods re expensive nd mke the growth of multiple tumors unrelistic for most lortories. Replcing these mterils with gr gel nd other commonly used sustnces could provide relile, prmeter-controlled source of invitro tumors. The cell line chosen for this experiment ws the T mmmry crcinom cell line produced in BALB/ cfc3h mice nd first grown t the Krmnos Cncer Institute y Fred Miller (To et l., 28). This cell line ws chosen due to its tumorigenicity nd invsivity. Furthermore, the T cell line is n idel cndidte A % FBS DMEM medi ws mde from 5mL Fetl Bovine Serum, 5mL Penicillin-Streptomycin solution, 5mL L-glutmine, nd ml Hyclone Dulecco s High Glucose Modified Egles Medium (DMEM). The T cell line ws otined from Dr. Dvid Zhroff s l t the University of Arknss. They were cultured in the % FBS DMEM medi nd incuted in humidified environment t 37 C nd wshed with Phosphte Buffered Sline (PBS) every two-three dys. Ech week, the cells were pssged nd two-three million cells were redistriuted to new flsk with fresh medi. Flt-ottomed pltes Ech 6mm flt-ottomed culture plte ws lined with ml of gr gel mde from 2mL of % FBS DMEM medi,.33ml of.8% Nole Agr, nd.67ml of sterile wter. After setting for ten minutes, mix of 2.67mL of the T cell solution nd.33ml of.8% Nole Agr ws dded on top of the gel. Once the gel set, 2mL of medi ws dded on top nd the pltes were incuted for two nights. The pltes were viewed under n opticl microscope every two or three dys for four weeks. Fresh medi ws dded to the pltes to provide new nutrient source for the cells. Round-ottom multiwell pltes One percent gr ws prepred from ml sterile wter nd.g Difco Nole Agr, then utoclved for fifteen minutes t 5 C to sterilize the solution. Fifty μl of gr ws pipetted into ech well of the first four rows (A-D) of 96 well ultr-low ttchment multiwell plte. The plte ws constntly rotted s the gel cooled to ensure n even lyer of gel coted the well while retining the curved ottom surfce. It ws then overlid with μl of % FBS DMEM medi to 5

3 prevent the gel from drying out nd llowed to incute overnight. 5, cells were dded to Row A, 3, were dded to Row B, 6, were dded to Row C, nd.2 million were dded to Row D. The % FBS DMEM medi tht contined the cells ws chnged every two to three dys to continue providing nutrients. Preprtion of the smples Multiple methods were used to prepre the smples for the THz spectroscopy nd imging system. Some of the flt-ottomed pltes were fixed using 2% prformldehyde solution while others were left s controls. Tissue Tek O.C.T. Compound (OCT) ws dded to oth smples in plstic form to ensure optiml cutting temperture once frozen. Both types of pltes were frozen nd kept t -2 C. Using Leic CM86 cryostt, the smples were sectioned t thickness of 2 µm. The round-ottomed multiwell plte ws frozen t -2 Celsius. Three smples with strting cell count of 6, cells were sectioned with thicknesses of µm, 5µm, nd µm nd exposed to the ir overnight to llow wter molecules to evporte from the OCT nd gel efore imging. Another smple with strting count of.2 million cells ws sliced to thickness of µm nd plced directly on the viewing plte, preventing evportion. A smple with strting cell count of 5, cells ws thwed, trnsferred directly to the viewing plte, nd dried. All smples were imged using the TPS Spectr 3 terhertz spectrometer from Terview nd nlyzed using the TVL Imging Suite (softwre pckge of the system). Figure 2. Photomicrogrphs of spheroids from fltottomed pltes. Spheroids were immersed within the gel nd imged t mgnifiction. demonstrtes lmost uniform reflection off the entire surfce of the smple. The reflection imging module of the THz system is shown in Figure. Round-ottom multiwell pltes Wells contining over 5, cells ppered crowded with no res of incresed cell concentrtion or spheroid formtion. The smples otined from the 6, wells were difficult to section nd the gel ppered very rittle nd frgmented. Upon slicing, the gel contining the smple would flke nd disintegrte s it rolled off the smple lock or cme into contct with the metl on the cryostt mchine. Thicker sections were tken to otin solid smple without flking. THz viewing showed no positive reflection where the tumor should hve een. The µm slice showed the sme reflection in the center of the OCT, where the tumor should hve een, s the Results Flt-ottomed pltes The spheroids grown in the flt-ottomed pltes grew to mximum size of 2 µm with mximum center of 75 µm (Fig. 2). They were completely encsed in the gr gel se nd were esily sectioned due to the gel. In order to otin THz imges, the spheroids were emedded in OCT compound. Imging these smples using the THz system did not show ny field significnt reflection from the spheroid grown in fltottomed pltes. As shown in Figure 3, the THz imge Figure 3. Cross section of T spheroids from fltottomed plte. The smple ws emedded in gr gel nd OCT compound. A 2µm thick section ws cut nd imged using the THz spectroscopy nd imging system. 55

4 c d.2..6 x-xis (mm).8 Figure 5. Cross section of T spheroid from 6, strting cells. The smple ws emedded in gr gel nd surrounded y OCT Compound. A µm thick section ws cut nd imged using the THz spectroscopy nd imging system. The yellow re, where the tumor should hve een, within the red re indictes lck of cncer cells x-xis (mm).8 Figure 6. Cross section of T spheroid from 6, strting cells. The smple ws emedded in gr gel nd surrounded y OCT Compound. A 5µm thick section ws cut nd imged using the THz spectroscopy nd imging system. The uniform reflectnce cross the smple indictes lck of cncer cells. 2.8 re tht hd only the viewing plte (Fig. 5). The sme ws the cse for the 5µm (Fig.6) nd µm (Fig. 7) slices. The right lue spot on the µm slice ws oserved s ule (Fig. 7). The smple from the well, eginning with.2 million cells, gin disintegrted during sectioning nd only showed single reflection off the center of the viewing plte. No spheroids or concentrtions of cells were present (Fig. 8). Wells with 5, strting cells produced spheroids centered in the middle with multiple tighter spheroids round the edges (Fig. 9). Trnsporttion of the smples to the viewing pltes showed the tumors were emedded into the gel, ut it is not cler how deep. The colors seen on the THz imges re ssigned ritrrily to est show the difference in reflections tht re given off ecuse the originl imge is lck nd white. THz spectroscopy showed difference in reflection etween the medi tht contined the smple nd the tumors s whole. In Figure, the medium lue re surrounding the righter lue shows negtive reflection, ut one with lower mgnitude thn the viewing plte. The rightest lue shows positive reflection while the drkest lue shows negtive Figure. Imges of the TPS Spectr 3 spectrometer ) shows the core system s whole, ) shows more detil of the reflection imging module, c) shows the plcement of smples within the viewing window, nd d) shows closeup of potentil smple. BIOLOGICAL SCIENCES: Scrlett-Mrie Acklin (mm)..6 x-xis.8 Figure 7. Cross section of T spheroid from 6, strting cells. The smple ws emedded in gr gel nd surrounded y OCT Compound. A µm thick section ws cut nd imged using the THz spectroscopy nd imging system. The uniform reflectnce cross the smple indictes lck of cncer cells, s the lue portion ws ule. 56

5 8 6 2 Figure 8. Cross section of T spheroid from,2, strting cells. The smple ws emedded in gr gel nd surrounded y OCT Compound. A thick section ws cut nd imged using the THz spectroscopy imging system. The single reflectnce in the center of the imge corresponds to only OCT compound nd indictes lck of tumor. reflection. The re of positive reflection corresponded to the re of high cell density on the microphotogrph nd the negtive reflection responded to the re of low density. The tumor mesured out 5mm in dimeter. Figure shows similr results with the centrl c Figure 9. Photomicrogrphs of spheroids t mgnifiction from round-ottomed multiwell plte. The cells nd spheroids were emedded into the gel se lyer nd the initil cell count ws 5,. Figures,, nd c represent the spheroids clusters tken t different position of the smple. Figure. THz cross sections of T spheroids from 5, strting cells, first smple. The smple ws emedded in gr gel nd processed in its entirety using the THz spectrometer nd imged t mgnifiction. Figure, shows positive reflection in the right lue re nd negtive reflection in the drkest lue re, corresponding to the re of highest density nd re of low density in the microphotogrph shown in Figure. The imge ws tken of the entire smple in petri dish. red re exhiiting positive reflection, the drkest lue re showing negtive reflection, nd the yellow re round it demonstrting negtive reflection with lower mgnitude thn the viewing plte. The red re correlted to the re of high density on the microphotogrph nd the drk lue correlted to the re of low density. Approximte size of the tumor ws 7mm in dimeter. 57

6 Figure. THz cross sections of T spheroids from 5, strting cells, second smple. The smple ws emedded in gr gel nd processed in its entirety using the THz spectrometer nd imged t mgnifiction. Figure, shows positive reflection in the red re nd negtive reflection in the drkest lue re, corresponding to the re of highest density nd re of low density in the microphotogrph shown in Figure. The imge ws tken of the entire smple in petri dish. Figure 2 lso supported the previous findings. The red nd lck res showed positive reflection, with lck hving higher mgnitude. The two lck res in the lrger spheroid corresponded to the split spheroid seen in Figure 2, lthough the orienttion did not completely correspond. The second, smller tumor ws lso seen in the microphotogrph nd mesured 2mm in dimeter while the lrger one mesured 5mm in dimeter. All THz imges presented in this work represent the pek of the electric field reflected from the smples expressed in ritrry units (.u.), which could e the positive pek or the negtive pek of the pulse. Discussion Using the round-ottom multiwell plte helped ggregte the T cells together nd gve them greter opportunities for cell-to-cell contct to form the primry tumor s well s the peripherl spheroids. The smple otined from the round-ottomed well ws 25 the size of the lrgest spheroid grown in the fltottomed pltes. However, the environment in the fltottomed plte ensured the tumor ws fully encsed in gel, s it would e in n ctul ptient. Growing in this environment is not n option for this project, though, ecuse the THz spectroscopy ws not le to produce results from the smple. The insignificnt reflection of the electric field shown on the THz imge is likely Figure 2. THz cross sections of T spheroids from 5, strting cells, third smple. The smple ws emedded in gr gel nd processed in its entirety using the THz spectrometer nd imged t mgnifiction. Figure, shows positive reflection in the red res nd even stronger positive reflection in the lck res, corresponding to the multiple res of thick cell density re in the microphotogrph shown in Figures nd c. The imge ws tken of the entire smple in petri dish. due to the spheroids eing too smll for viewing, or the section could hve een too thin leding to desicction nd loss of smple. In the round-ottom environment, the uniform dispersl of cells strting with more thn 5, cells suggests there is n upper limit to the cell count tht cn grow in multiwell plte of this size. Potentil cuses re lck of spce, lower nutrient:cell rtio, nd limited ccess to the nutrients contined in the medi. In order to remove the entire smple from the well nd ensure ll prts of the tumor were removed s whole; freezing initilly seemed to e the est option ecuse it is lso required for sectioning. However, the gr gel ecme too rittle t the -2 C temperture in which it is sectioned, nd prevented qulity smple of the ctul tumor from eing included with the OCT Compound in the smple slice. This is shown in Figures 5-7 where the OCT produced electric field reflection different from tht of the viewing plte, ut the center of the plte hd the sme reflection s the plte. Additionl smples were plced onto different mteril, polyurethne, to c 58

7 ensure this ws not the result of the smple hving refrctive index equl to tht of the originl glss slide. Figure 8 lso supports the concept tht the wells cnnot hold tht mny cells s it produces uniform electric field reflection cross the entire slide. Trnsferring the smple directly to the viewing plte corrected this prolem while still differentiting etween the medi nd the T cells nd eliminted the need for the OCT compound. The resolution of the THz system ws not le to distinguish etween the entire smple nd the tighter spheroids tht were ttched to the outer edge of the tumor. This would e of less significnce when deling with ptient with rest cncer ecuse the tumor would e size lrge enough to e detected using the THz system. Microphotogrphs of the spheroids from the 5, wells strengthen the findings of the THz imging. Figure ligns with nd shows higher cell concentrtion nd tumor height on the left of the imge. More cells re seen on the right, with much lower density in etween. Figures nd lso correspond to ech other, showing decresed tumor thickness on the ottom left corner of the smple. Finlly, Figures 2 nd 2c illustrte the results otined in 2. The microphotogrph revels the two drk res within the tumor s res of greter tumor thickness nd gives more detil to the smller, seprted spheroid. Bsed on the results, prticulrly Figure 2c tht shows seprte smller spheroid tht grew in ddition to the primry tumor, THz imging nd spectroscopy is vile method for detecting rest cncer tissues of ptients with tumors 2mm or lrger. This size is much lower thn the current medin detectle size, 7.5mm (Michelson et l., 23). For future reserch, it is recommended plting 5, T cells in 96 well multiwell plte with se lyer of gr mintining the rounded shpe. To prepre nd imge the smple using the THz spectrometer, the smple should e trnsferred directly to the viewing plte without prior freezing nd ll wter should e evported overnight. The initil gol of this project ws to grow rest tumors in vitro tht mimic in vivo tumors in size nd encsement in flexile sustnce. While the tumors were limited in size, the results reveled new methodology for growing tumors for THz spectroscopy t lower cost tht could e replicted on lrger scle. Works Cited. Alerts, B., Johnson, A., Lewis, J., Rff, M., Roerts, K., & Wlter, P. (22). Moleculr Biology of the Cell. New York. Grlnd Science. 2. Americn Cncer Society (25). Cncer Fct & Figures. Americn Cncer Society. Mrch Bowmn, T. C., El-Shenwee, M., & Cmpell, L. K. (25). Experimentl terhertz z-scn imging of three-dimensionl prffin emedded rest cncer tissue. Proceedings of IEEE MTT-S Interntionl Microwve Symposium, Phoenix, AZ, USA.. Bowmn, T. C., El-Shenwee, M. & Cmpell, L. K. (25). Terhertz imging of excised rest tumor tissue on prffin sections. IEEE Trnsctions on Antenns nd Propgtion, 63, Brestcncer.org (25), Surgicl Mrgins. Mrch Burford, N., El-Shenwee, M, O Nel, C.B. & Olejniczk, K. J. (2). Terhertz imging for nondestructive evlution of pckged power electronic devices. Interntionl Journl of Emerging Technology nd Advnced Engineering,, Dimri, M., Nrmur, M., Dun, L., Chen, J., Orteg-Cv, C., Chen, G., Goswmi, R., Fernndes, N., Go, Q., Dimri, G. P. Bnd, V., & Bnd, H. (27). Modeling rest cncerssocited c-src nd EGFR overexpression in humn MECS: c-src nd EGFR coopertively promote errnt threedimensionl cinr structure nd invsive ehvior. Cncer Reserch, 67, Fntozzi, A. & Gerhrd, C. (26). Mouse models of rest cncer metstsis. Brest Cncer Reserch, 8, Hssn, A. M., nd El-Shenwee, M. (2). Electromgnetic techniques for rest cncer detection. IEEE Reviews in Biomedicl Engineering,, Huynh, P., Jrolimek, A., & Susnne, D. The flsenegtive mmmogrm. (998). RdioGrphics, 8,

8 . Jepsen, P., Jcosen, R., & Keiding, S. (996). Genertion nd detection of terhertz pulses from ised semiconductor ntenns. Journl of the Opticl Society of Americ B, 3, Michelson, J., Stij, S., Moore, R., Weer, G., Hlpern, E., Grlnd, A., Kopns, D. B. & Hughes, K. (23). Estimtes of the sizes t which rest cncers ecome detectle on mmmogrphic nd clinicl grounds. Journl of Women s Imging, 5, Office of Environmentl Helth nd Sfety. (25). Biologicl effects of ionizing rdition. Princeton University. 9 Apr. 2. We. 29 Mr Pleijhuis, R. G., Grflnd, M., de Vries, J., Brt, J., de Jong, J. S., vn Dm, G. M. (29). Otining dequte surgicl mrgins in rest conserving therpy for ptients with erlystge rest cncer: Current modlities nd future directions. Ann. Surg. Oncol., 6, Pulski, B., & Ostrnd-Rosenerg, S. (2). Mouse T rest tumor model. Current Protocols in Immunology, 39, Sto, H., Goto, M., & Shigetoshi, H. (977). Growth ehvior of scites tumor cells in three-dimensionl gr culture. Tohoku Journl of Experimentl Medicine, 22, To, K., Fng, M., Alroy, J. & Shgin, G. G. (28). Imgle T model for the study of lte stge rest cncer. BMC Cncer, 8, Weigelt, B., Peterse, J., & Lur, J.V. (25). Brest cncer metstsis: mrkers nd models. Nture Reviews, 5, Wilmink, G., & Grundt, J. (2). Current stte of reserch on iologicl effects of terhertz rdition. Journl of Infrred, Millimeter nd Terhertz Wves, 32,