cell migration is critically important 
 in diverse physiological 
 contexts such as development , immune response , and wound healing , 
 and pathological contexts such as cancer . for example , cell migration 
 allows cancer cells to move away from the tumor , enter and exit blood 
 and lymph vessels , and migrate to secondary tumor sites , in a process 
 called metastasis . metastasis is one of the main 
 causes of mortality in cancer patients and consequently diagnostics 
 aimed at predicting or therapeutic interventions aimed at halting 
 metastasis are attractive . cues in 
 the extracellular environment that direct migration are numerous and 
 can either cooperate or compete to direct migration . these cues include gradients in growth factors or extracellular 
 matrix ( ecm ) concentration , mechanical properties , or electric field . gradients often bias migration toward targets and are thus monodirectional . 
 aligned fibers composed of ecm also direct cell migration . however , 
 aligned fibers bias migration along their long axis and are thus bidirectional . contact guidance specifically impacts wound healing , immune response , neuronal 
 development and repair , and cancer metastasis . however , even though cell migration can be directed 
 through several different cues , the fundamental processes carried 
 out during cell migration appear to be conserved . cell migration proceeds in steps : protrusion , adhesion , 
 and contraction , 
 which result in traction force generation and tail retraction . in 3d environments the 
 cell senses contact guidance cues via adhesion between receptors such 
 as integrins and the fibers . much of this work has been conducted 
 on ridges that mimic ecm fibers showing that contact guidance cues 
 act to align focal adhesions and the actin cytoskeleton , resulting in directional migration . along with focal adhesions and 
 the actin cytoskeleton , the ability of cells to transmit force seems 
 to play a role as decreasing contractility seems to diminish contact 
 guidance fidelity on microcontact printed lines of collagen type i. migrational steps are organized differently 
 in different cells , resulting in a developing hypothesis that cells 
 adopt different modes of migration . single 
 cell modes include the amoeboid , mesenchymal , blebbing , lobopodial , and 
 cytoskeletal independent modes of migration . of these these cells are also dependent on myosin contractility 
 to squeeze through pores , but tend to not bind the ecm strongly . these cells 
 are less dependent on myosin contractility as they can degrade matrix 
 to generate larger pores , but tend to bind the matrix more strongly . 
 whether amoeboid or mesenchymal , cells must still interact with the 
 ecm , even if weakly or nonspecifically . the ecm that surrounds the 
 tumor is often composed of a dense but porous network of entangled 
 and perhaps cross - linked collagen type i. collagen type i is a heterotrimer that assembles into fibrils 
 with 
 tightly regulated ultrastructural characteristics such as d - periodic 
 banding . this structure is a hallmark 
 of biologically relevant collagen fibrils and is critical for contact 
 guidance . collagen fibrils can be aligned 
 and bundled into fibers through contractility mediated mechanisms 
 in some tissues . in particular , the tissue surrounding 
 breast tumors appears to undergo a dramatic change , whereby fibers 
 are no longer oriented parallel to the tumor margin , but are oriented 
 perpendicularly to the tumor margin . this 
 facilitates directed migration of cancer cells to the surrounding 
 tissue during invasion and metastasis . additionally , the density and 
 mechanical properties of collagen ecm has been suggested as a good 
 indicator of future invasion and metastasis . consequently , 
 to better understand fundamental cell biology and design better diagnostic 
 strategies , it is important to understand how cell migration is quantitatively 
 altered in response to collagen fiber characteristics such as degree 
 of alignment , diameter , density , and mechanical properties , as well 
 as among different cells with intrinsically different migrational 
 modes . this requires using systems where collagen fibril ( or fiber ) 
 characteristics can be altered and assessing cell migration behavior 
 across several different cell lines . the past 10 years have 
 seen a dramatic increase in the engineering 
 approaches by which to fabricate fiber networks with specific fiber 
 characteristics . in particular , several techniques have emerged to 
 generate aligned fibers or fiber - like entities . these methods include 
 fabricating ridges , electrospinning , fiber drawing , epitaxial 
 growth , microcontact printing , flow - based alignment , and magnetic bead alignment . surprisingly , 
 these approaches have not been fully utilized to understand contact 
 guidance . here we focus on the elegant and simple approach of epitaxially 
 growing collagen type i ( hereafter referred to as collagen ) fibrils 
 on mica to generate fibril structures with different organizational 
 characteristics . collagen fibril assembly is governed 
 by collagen  collagen and collagen  mica electrostatic 
 interactions . the interaction with mica is 
 due in part to potassium ions that remain on the surface after cleavage . solution phase conditions such as the collagen 
 concentration and incubation time determine fibril size and density , the ph of the solution determines fibril density and alignment , and the ionic species determine the fibril density , alignment , and 
 presence of d - banding . because potassium ions seem to be important in initiating 
 d - banding , others have examined different potassium ion concentrations , 
 and they appear to regulate fibril size , density , and alignment . finally , muscovite mica orders collagen differently than phlogopite 
 mica . the geometry of epitaxially grown collagen 
 fibrils resembles a thin tape as opposed to a cylinder as seen in 
 vivo . however , there are several advantages to this approach over 
 other approaches described above , including the ease in forming a 
 diverse set of structures and efficiency in forming d - bands with similar 
 spacing as those seen in collagen fibrils in vivo . while these substrates 
 have been used to assess the effect of d - banding on fibroblast contact 
 guidance and the coupling of adhesion 
 and proteinase activity , they have not 
 been used to assess contact guidance across different collagen structures 
 and among different cell types . in this paper , we generated 
 several different organizational patterns 
 of collagen and assessed how cell morphology , orientation , and contact 
 guidance behavior depended on the collagen organization . disorganized 
 collagen fibrils and physically adsorbed collagen allowed cells to 
 spread , but did not direct migration . we examined this behavior in 
 two different breast cancer cell types and found profound differences . mda - mb-231 cells which migrate with a mesenchymal mode of migration 
 and which exert high traction force were highly directional , but mtln3 
 cells which migrate with an amoeboid mode of migration and which exert 
 lower traction force were not directional at all , even though they 
 spread and migrated well on the aligned fibrils . this demonstrates 
 that different contact guidance cues can bias cell migration differently 
 and the fidelity of contact guidance is highly dependent on cell type , 
 suggesting that the ecm alignment is a permissive cue for contact 
 guidance , but requires a cell to have certain properties in order 
 to interpret that cue . collagen fibrils assembled on mica offer an 
 interesting platform on which to exquisitely tune contact guidance 
 signals and examine cellular responses at the level of migration . a rat mammary basal adenocarcinoma cell 
 line ( mtln3 , jeffrey e. segall , albert 
 einstein college of medicine ) was maintained in subculturing media 
 at 37 c in 5% co2 . subculturing media for mtln3 cells 
 was mem ( life technologies , grand island , ny , usa ) containing 
 5% fetal bovine serum ( life technologies ) and 1% penicillin  streptomycin 
 ( life technologies ) . a human mammary basal / claudin low carcinoma cell line ( mda - mb-231 , atcc , manassas , 
 va , usa ) subculturing media for mda - mb-231 cells was dmem ( life 
 technologies ) containing 10% fetal bovine serum ( life technologies ) 
 and 1% penicillin imaging 
 media was the same as the subculturing media , with the exception that 
 no phenol red was included and that 12 mm hepes ( sigma - aldrich , st . high concentration non - pepsin - treated 
 rat tail collagen type i ( bd bioscience , hampton , new hampshire , usa ) 
 was used . freshly and uniformly 
 cleaving mica is a required step in assembling reproducible collagen 
 fibril fields . a 15 mm  15 mm piece of muscovite mica ( highest 
 grade vi , ted pella , redding , ca , usa ) was freshly cleaved using tape . collagen type i was diluted in the buffer solution to the proper concentration 
 and the buffer solution was incubated on the mica at room temperature . after incubation the collagen solution was washed with distilled water , 
 the mica was laid against the edge of a tissue culture dish , and the 
 mica was allowed to dry overnight and used the next day . buffer solution 1 consisted of 50 
 mm tris - hcl ( fisher scientific , hampton , new hampshire , usa ) and 200 
 mm kcl ( fisher scientific ) at ph 9.2 . buffer solution 2 consisted 
 of 50 mm tris - hcl and 200 mm kcl at ph 7.5 . buffer solution 3 consisted 
 of 50 mm citric acid - sodium citrate ( fisher scientific ) and 200 mm 
 kcl at ph 4.3 . buffer solution 4 consisted of 50 mm citric acid - sodium 
 citrate and 0 mm kcl at ph 4.2 . a dimension 3100 scanning probe 
 microscope with nanoscope iv controller 
 ( veeco metrology , llc , santa barbara , ca ) was utilized to obtain height 
 images of collagen fibrils on mica . imaging was conducted in tapping 
 mode using silicon tesp7 afm tips ( veeco metrology , llc , santa barbara , 
 ca ) with a spring constant of 79 n / m and resonance frequency 
 of 269 khz . roughness of type i collagen fibrils was calculated 
 by computing the standard deviation of the height over a 5 m 
  5 m scan area . fast fourier transform ( fft ) aspect ratio 
 processing steps are shown in supporting information 
 figure 1 and all steps were carried out in imagej . this fft operation translates 
 spatial information into frequency information and is thus useful 
 in quantifying repeating patterns and length scales . this fft image 
 was binarized based on a threshold that was two standard deviations 
 higher than the fft image average , resulting in a shape centered at 
 the origin . several serial image processing steps were conducted in 
 imagej in order to eliminate small objects and fill holes in the dominant 
 shape centered at the origin . the following processing steps were 
 used in order : fill holes ( 4 connected background elements ) , erode , 
 dilate , open , and close ( structured element for erosion , dilation , 
 opening and closing is a 3  3 matrix ) . finally , an ellipse was fit to the 
 zero order signal using the analyze particles function in imagej , 
 which was always the largest continuous shape . the aspect ratio was 
 defined as the long axis divided by the short axis of the ellipse . fibril height was calculated by fitting the height distribution with 
 a gaussian function and determining the peak width at 30% of the maximum 
 height . under this criterion the thickness of the unpolymerized collagen 
 was roughly 1.5 nm , which matches well with the known diameter of 
 the collagen heterotrimer . fibril height could not be unambiguously 
 determined given the high density of the fibril fields and some uncertainty 
 as to whether the afm tip could at times penetrate to the mica layer 
 below . fibril width and length were measured manually using imagej . 
 while there is high confidence in fibril width , fibril length is a 
 bit more difficult to measure given some ambiguity in where fibrils 
 start and stop . mda - mb-231 cells 
 were incubated for 
 1 h and mtln3 cells were incubated for 12 h on organized type i collagen 
 substrates in imaging media . mica with type i collagen fibrils and 
 cells attached to the fibrils were inverted onto two strips of double - sided 
 tape attached to a microscope slide to generate a flow chamber . chambers 
 were imaged by phase contrast microscopy on a heated stage at 37 c 
 every 2 min for 12 h. images were captured at 10 ( na 0.50 , nikon ) 
 with a charge - coupled device ( coolsnap hq2 , photometrics ) attached 
 to an inverted microscope ( ti - e , nikon ) that was driven by manager . cell speed and directionality 
 were calculated over a time lag of 2 min averaged over 12 h as described 
 in a previous paper . mda - mb-231 cells were incubated 
 for 4 h and mtln3 cells were incubated for 12 h on organized type 
 i collagen , fixed with 4% paraformaldehyde , permeabilized with 0.5% 
 triton - x , and stained for f - actin , a cytoskeletal protein and paxillin , 
 an adhesion protein . f - actin was stained using alexa 488-phalloidin 
 ( life technologies ) and paxillin was stained using mouse - anti - paxillin 
 antibody ( 349 , bd biosciences ) and a donkey - anti - mouse cy3 antibody 
 ( jackson immuno research ) . fixed and stained cells were imaged by 
 epifluorescence using a 60 oil objective ( na 1.49 , nikon ) on 
 the same microscope as described above . aspect ratio for each cell 
 was calculated by dividing the length of each cell by its width . of 
 the cells that were adequately elongated ( aspect ratio 1.3 ) , 
 the orientation direction was calculated along the long axis of the 
 cell . orientation distributions across experimental replicates over 
 several days were aligned and the dominant peak was set at 30 , 
 given that the fibril orientation and cell orientation were not readily 
 imaged simultaneously . furthermore , orientation distributions were 
 fitted by double gaussians governed by the following equation:1where f is 
 the frequency fraction and  is the orientation angle in deg . 
 the second and fourth term arise in order to ensure that the gaussian 
 is propagated periodically . for wide distributions ( large b1 and b2 ) , the left 
 tail of the gaussian at 30 contributes to fractions around 180 
 and the right tail of the gaussian at 90 contributes to fractions 
 around 0. confidence intervals of 95% were calculated using 
 the nlparci function in matlab ( mathworks ) , which uses the fitting 
 residuals and the jacobian of the fitting equation to estimate the 
 95% confidence intervals on fitting parameters . a peak was assigned 
 only if the confidence interval of a1 or a2 was greater than zero and if that of b1 or b2 was less 
 than 30. wide distributions are less likely to be peaks . if 
 either of these criteria was not achieved , fits to either a single 
 gaussian ( terms 1 and 2 of eq 1 ) or a constant 
 value were used . determining 
 the contact guidance fidelity of migrating cells requires tight control 
 over the organization of the ecm . furthermore , the fibril 
 characteristics can be controlled by solution phase properties such 
 as ph , ionic strength , ion species , incubation time , and collagen 
 concentration . in this paper , we characterize collagen organization 
 using atomic force microscopy ( afm ) under six conditions ( figure 1a  f ) . conditions were chosen to yield a diversity 
 of collagen organizational patterns , including highly aligned fibrils 
 of large width and height ( figure 1a ) , multidirectional 
 fibrils of intermediate width and height ( figure 1b ) , poorly aligned fibrils of small width and height ( figure 1c and d ) , and punctate spots ( figure 1e ) . as seen previously , solutions with high ph and/or 
 long incubation times / high collagen concentrations produced the largest 
 fibrils ( figure 1g , h ; see a and b ) . solutions 
 with intermediate to low ph coupled with short incubation times / low 
 collagen concentrations produced smaller fibrils ( figure 1g , h ; see c and d ) . finally , solutions with low ph 
 with no potassium ions inhibited fibril formation . the degree of fibril 
 alignment was quantified by examining the fast fourier transform ( fft ) 
 of the image and measuring the aspect ratio of the zero order signal 
 after image processing as described in the experimental section ( supporting information figure 1 ) . randomly organized fields ( e and f ) or fibril fields with dominant 
 angles that are nearly perpendicular ( b ) should have an aspect ratio 
 close to 1 , and highly aligned fibrils ( a ) should have a large aspect 
 ratio ( figure 1i ) . the highly organized , large 
 collagen fibrils show a characteristic d - banding along their length , 
 producing stripes in the fft oriented perpendicularly to the fibril 
 orientation ( supporting information figure 1a ) . consequently , the long axis of the high - intensity zero - order signal 
 is perpendicular to the orientation axis of the fibrils . in addition 
 to the fft aspect ratio , the roughness of the surface was calculated 
 showing that fibrils of larger diameter ( a and b ) were rougher than 
 the smaller fibrils ( c and d ) , collagen pucta ( e ) , or bare mica ( f ) 
 ( figure 1i ) . comparison of the roughness of 
 condition a with the roughness of condition c , d , or e using a two - tailed , 
 unequal variance student s t - test yielded p values of less than 0.05 . comparison of the roughness 
 of condition b with the roughness of condition c , d , or e yielded p values of less than 0.05 , except for e which yielded a p value of 0.095 . finally , when roughnesses of a and b as 
 well as c and d were grouped together the p values 
 for comparison in roughness between groups ab and cd or e were both 
 less than 0.05 . all conditions were at least twice as rough as bare 
 mica . given this surface characterization of the collagen organization , 
 we were interested in determining cell adhesion and migration behavior 
 on the different collagen organizational patterns . characterization of collagen 
 organization on freshly cleaved mica 
 using atomic force microscopy ( afm ) under six conditions : ( a ) ph 9.2 , 
 200 mm kcl , 10 g / ml collagen , 1.5 h incubation time ; ( b ) ph 
 7.5 , 200 mm kcl , 100 g / ml collagen , 3 h incubation time ; ( c ) 
 ph 7.5 , 200 mm kcl , 100 g / ml collagen , 20 min incubation time ; 
 ( d ) ph 4.3 , 200 mm kcl , 10 g / ml collagen , 20 min incubation 
 time ; ( e ) ph 4.2 , 0 mm kcl , 100 g / ml collagen , 3 h incubation 
 time ; and ( f ) bare mica . top : 5 m  5 m scan ( calibration 
 bar length = 1 m ) . bottom : 1 m  1 m enlarged 
 region outlined in the square above ( calibration bar length = 200 
 nm ) . each 
 data point was generated from a sample image from one independent 
 collagen deposition experiment . error bars are 95% confidence intervals 
 for fibril length and width were determined from measuring > 10 
 fibrils 
 per image . ( i ) fft aspect ratio and surface roughness . a : black squares , 
 b : black circles , c : gray circles , d : gray triangles , e : open triangles , 
 and f : open diamond . these cells adhere and migrate on collagen fibrils , are 
 contractile , and adopt a mesenchymal mode of migration , which is characterized 
 by high proteinase activity coupled with adhesion to the ecm . mda - mb-231 
 cells were plated on the six different substrates characterized in 
 figure 1 and were fixed and stained for both 
 f - actin , a cytoskeletal protein , and paxillin , an adhesion protein . cells were spread and elongated on the highly aligned fibrils of large 
 width and height ( figure 2a ) . cells were spread , 
 but not elongated on other fibril organizations and punctate collagen 
 ( figure 2b  e ) . finally , cells did not 
 spread on the bare mica ( figure 2f ) . high resolution 
 images show that focal adhesions and bundles of f - actin are generated 
 on any substrate with collagen . aligned collagen fibrils of large 
 width and height result in elongation of both focal adhesions and 
 f - actin bundles ( figure 3 ) . the cell morphology 
 was quantified by calculating an aspect ratio of the cell from the 
 length and width measurements and agreed with the qualitative observations 
 ( figure 2 g ) . cell aspect ratios were divided 
 into groups including cells that were elongated ( white ) , slightly 
 elongated ( gray ) , not elongated , but spread ( dark gray ) , and not spread 
 ( darkest gray ) . the population of cells was not homogeneous , so the 
 fraction of cells in each aspect ratio group was plotted and showed 
 a consistent trend of a shift in the distribution from elongated , 
 spread cells , to unspread cells as the organization of the collagen 
 decreased ( a  f ) ( figure 2h ) . morphology 
 and orientation of mda - mb-231 cells under six organized 
 collagen assembly conditions . ( g ) cell aspect 
 ratio was calculated for cells by examining f - actin images for conditions 
 shown in figure 1a  f ( a. ( ndays = 5 and ncells = 909 ) , 
 b. ( ndays = 3 and ncells = 550 ) , c. ( ndays = 3 and ncells = 427 ) , d. ( ndays = 3 and ncells = 316 ) , e. ( ndays = 3 and ncells = 526 ) , 
 f. ( ndays = 3 and ncells = 165 ) ) . h. the frequency fraction of cells within each aspect ratio range 
 denoted with the same gray levels as shown in g and for conditions 
 shown in figure 1a  f . ( i  m ) angle 
 distributions of cells for conditions shown in figure 1a  f with aspect ratios > 1.3 ( a. ( ndays = 4 and ncells = 575 ) , b. ( ndays = 2 and ncells = 362 ) , c. ( ndays = 2 and ncells = 294 ) , d. ( ndays = 2 and ncells = 113 ) , 
 e. ( ndays = 2 and ncells = 294 ) ) . morphology and protein localization of mda - mb-231 cells under six 
 differently organized collagen substrates . ( a  f ) f 
 and stained for top : f - actin and bottom : a focal adhesion marker , 
 paxillin . calibration bar length = 30 m . high cell elongation does not necessarily indicate high cell 
 alignment 
 with the fibrils or a high degree of contact guidance , so the orientation 
 distributions of the cells were calculated as well . the highly aligned 
 fibrils of large width and height generated two peaks , which were 
 offset by 60 ( figure 2i ) . while across 
 one coverslip the alignment direction is fairly homogeneous , other 
 less frequent directions are seen ( figure 4a ) . this contrasted with the no fibril condition that showed random 
 alignment ( figure 4b ) . however , others have shown 
 that collagen fibril orientation is set by surface charge polarity established by parallel and alternating lines 
 of silicon oxide above and below the surface plane in the [ 1 1 0 ] 
 direction . when mica is incompletely 
 cleaved , steps corresponding to a different layer of mica can remain 
 on the surface at some places on the sample . these two different mica 
 layers might have a difference in the orientation direction of these 
 lines of silicon oxide above and below the surface plane , leading 
 to alternative collagen fibril and cell orientations at 60 
 over large length scales ( 1000 m ) . the multidirectional 
 fibrils of large width and height display multiple directions , even 
 on small length scales ( 5 m ) , so the orientation peaks 
 are much less pronounced ( figure 2j ) . the other 
 fibril orientations show solitary peaks ( figure 2k ) or no peaks at all ( figure 2l ) . we quantified the correlation between either the cell aspect 
 ratio or cell orientation peak height and fft aspect ratio or surface 
 roughness using the pearson correlation coefficient . cell aspect ratio 
 correlated better with surface roughness ( r = 0.77 ) 
 than fft aspect ratio ( r = 0.099 ) . on the other hand , cell orientation peak height correlated slightly better with fft aspect 
 ratio ( r = 0.37 ) than surface roughness ( r = 0.33 ) . these data along with those in figure 2 indicate that the aligned collagen fibrils of large 
 width and height , and to a lesser extent the multidirectional collagen 
 fibrils of large width and height are best able to generate elongation 
 and orientation in mda - mb-231 cells , suggesting they are better contact 
 guidance substrates . given this , we examined the contact guidance 
 fidelity directly by measuring the directionality of mda - mb-231 cells 
 migrating on these organizational patterns of collagen . mda - mb-231 
 cells were plated on substrates shown in figure 1a and b , imaged over 12 h ( figure 5a ) and 
 cell nuclei were tracked . cell directionality was calculated using 
 the following equation:2where n is the number of 
 time intervals contained in an individual cell track and i is the angle of the cell movement with respect 
 to the collagen fibrils . the directionality essentially quantifies 
 the projection of cell displacement along the long axis of the fibrils , 
 thus giving a good representation of the contact guidance fidelity . a directionality of 1 defines a situation where a cell only moves 
 in two directions along the fibril ( 0 and 180 ) . a directionality 
 of 0 defines a situation where a cell moves randomly or at a 45 
 with respect to the fibrils . finally , a directionality of 1 
 defines a situation where a cell moves perpendicularly with respect 
 to a fibril ( 90 ) . mda - mb-231 cells show high directionality 
 on aligned collagen fibrils of large width and height and a lower 
 directionality on the multidirectional collagen fibrils of large width 
 and height ( figure 5 ) . mda - mb-231 cells do 
 not directionally migrate on unpolymerized collagen substrates ( figure 5 ) . multidirectional collagen organization 
 resulted in high speed , whereas aligned collagen organization resulted 
 in lower speed ( figure 5b ) . cell speed on unpolymerized 
 collagen substrates was low and similar to that on the aligned collagen 
 fibrils ( figure 5b ) . the migrational behavior 
 on collagen fibrils agrees with many studies showing that directional 
 persistence is often inversely dependent on cell migration speed . this suggests that mda - mb-231 cells are sensitive 
 to aligned collagen fibrils , leading to high fidelity contact guidance . this is important given that in vivo data indicate the presence of 
 aligned collagen fibrils in the tumor microenvironment in breast cancer . cell orientation 
 of mda - mb-231 cells from images taken on different 
 areas of the epitaxially grown collagen for dense monodirectional 
 fibrils ( figure 1a , ph 9.2 , 200 mm kcl , 10 
 g / ml collagen , 1.5 h incubation time ) and no fibrils ( figure 1e , ph 4.2 , 0 mm kcl , 100 g / ml collagen , 3 
 h incubation ) . the coverslip is roughly 25 mm  25 mm and each 
 image field is roughly 0.4 mm  0.4 mm . the arrow direction indicates 
 the average cell orientation direction in an image field and the arrow 
 weight is inversely proportional to the standard deviation of directions 
 in an image field . a large standard deviation is denoted by a light 
 weight and a small standard deviation is denoted by a heavy weight . for random cell orientation angles , the average orientation is 90 
 ( pointing upward ) and the line weight approaches zero . migration behavior of mda - mb-231 cells on three different 
 organizational 
 patterns of epitaxially grown collagen : aligned collagen fibrils of 
 large width and height ( figure 1a , ph 9.2 , 
 200 mm kcl , 10 g / ml collagen , 1.5 h incubation time ) , multidirectional 
 collagen fibrils of large width and height ( figure 1b , ph 7.5 , 200 mm kcl , 100 g / ml collagen , 3 h incubation 
 time ) , and unpolymerized collagen ( figure 1e , ph 4.2 , 0 mm kcl , 100 g / ml collagen , 3 h incubation time ) . ( a ) montage of phase contrast images of mda - mb-231 cells at different 
 timepoints during migration . ( b ) left : directionality and right : cell migration speed of mda - mb-231 
 cells . we were interested in whether this contact 
 guidance behavior was universal or restricted to this one cell type , 
 so we performed similar assays using the mtln3 cell line , another 
 commonly used breast cancer cell line . both mda - mb-231 and mtln3 cells 
 are basal or basal - like cells and adhere and migrate on collagen ; 
 however , there are several differences . first , mda - mb-231 cells adopt 
 more of a mesenchymal mode of migration in 3d environments compared 
 to the amoeboid mode of migration that mtln3 cells adopt . in 2d environments , mtln3 cells adopt a fan shaped morphology , whereas 
 mda - mb-231 cells are more spindle - like . finally , mda - mb-231 cells exert roughly 10-fold higher traction 
 forces than mtln3 cells , resulting in more stable focal adhesions 
 and f - actin bundles and slower 2d migrational speeds . mtln3 cells generated focal adhesions , 
 assembled f - actin bundles , spread , and migrated on different organizational 
 patterns of collagen ( figures 6 and 7 ) . however , mtln3 cells did not elongate as readily 
 as mda - mb-231 cells ( figure 2 g and h compared 
 to figure 6a and b ) and showed no peak in cell 
 orientation on highly aligned fibrils ( figure 2i compared figure 6c ) . however , two peaks 
 did occur on multidirectional fibrils ( figure 2j compared with figure 6d ) . mtln3 cells migrated 
 more rapidly than mda - mb-231 cells on aligned fibrils , but slightly 
 more slowly on multidirectional fibrils ( figure 5b compared to figure 7b ) . finally , mtln3 cells did 
 not interpret the aligned fibrils as a contact guidance cue ( figure 7b ) . mda - mb-231 cells had a much higher directionality 
 than mtln3 cells under both fibril organizational conditions ( figure 5 compared to figure 7 ) . this 
 is interesting , given that mtln3 cells migrate directionally on microcontact 
 printed lines of collagen on mica ( supporting 
 information figure 2 ) and glass . first , 
 microcontact printed collagen is heterotrimeric and not assembled 
 into collagen fibrils . it is possible that certain contact guidance 
 sites for mtln3 cells are masked when collagen assembles into fibrils . second , the lines of collagen are sparser than the collagen fibrils 
 used here and separated by less adhesive areas . when mtln3 cells are 
 faced with nonadhesive areas as in the microcontact printed lines , morphology and orientation 
 of mtln-3 cells under two organized 
 collagen conditions : aligned collagen fibrils of large width and height 
 ( figure 1a , ph 9.2 , 200 mm kcl , 10 g / ml 
 collagen , 1.5 h incubation time ) and multidirectional collagen fibrils 
 of large width and height ( figure 1b , ph 7.5 , 
 200 mm kcl , 100 g / ml collagen , 3 h incubation time ) . ( a ) cell 
 aspect ratio was calculated for cells by examining f - actin images 
 for conditions shown in figure 1a and b ( a. 
 ( ndays = 5 and ncells = 940 ) and b. ( ndays = 2 
 and ncells = 242 ) ) . ( b ) frequency fraction of cells 
 within each aspect ratio range denoted with the same gray levels as 
 shown in a. ( c , d ) angle distributions of cells on for conditions shown 
 in figure 1a , b with aspect ratios of > 1.3 
 ( a. 
 ( ndays = 5 and ncells = 468 ) and b. ( ndays = 2 
 and ncells = 94 ) ) . ( e ) mtln3 cells 
 were plated on aligned fibrils of large width and height ( figure 1a , ph 9.2 , 200 mm kcl , 10 g / ml collagen , 
 1.5 h incubation time ) and multidirectional fibrils of large width 
 and height ( figure 1b , ph 7.5 , 200 mm kcl , 
 100 g / ml collagen , 3 h incubation time ) and stained for left : 
 a focal adhesion marker , paxillin , and right : f - actin . . migration behavior of mtln3 cells on two different organizational 
 patterns of epitaxially grown collagen : aligned collagen fibrils of 
 large width and height ( figure 1a , ph 9.2 , 
 200 mm kcl , 10 g / ml collagen , 1.5 h incubation time ) and multidirectional 
 collagen fibrils of large width and height ( figure 1b , ph 7.5 , 200 mm kcl , 100 g / ml collagen , 3 h incubation 
 time ) . ( a ) montage of phase contrast images of mtln3 cells at different 
 time points during migration . ( b ) left : directionality and right : cell migration speed of mtln3 
 cells . error bars are 95% confidence intervals . several possibilities could explain 
 the difference in contact guidance 
 fidelity between mda - mb-231 cells and mtln3 cells . first , it is possible 
 that altering the traction forces exerted by the cells on the substrate 
 might change the directional fidelity . traction force could act to 
 align focal adhesions , therefore by increasing the traction force 
 in mtln3 cells , one might make more directional cells and by decreasing 
 the traction force in mda - mb-231 cells , one might make less directional 
 cells . second , it is possible that receptor expression could be different 
 among the two cells leading to differences in directional fidelity . these experiments were conducted in serum , where multiple ecm proteins 
 such as fibronectin and vitronectin can bind to the collagen . perhaps 
 differences in the usage of 21 ( collagen ) integrins vs 51 and 
 v3 ( fibronectin and vitronectin ) 
 integrins might mediate this response . first , rho and its downstream 
 myosin light chain kinase , rock , are indeed important to contact guidance 
 in 3d collagen matrices . however , this importance is related to the 
 role of rho and rock in collagen reorganization rather than a role 
 in intrinsic ability to sense contact guidance cues . indeed , inhibiting 
 rho and rock however , it is possible that rho and rock regulate 
 intrinsic contact guidance differently between 2d and 3d , making it 
 a possible candidate to explain the differences between these cell 
 types . higher rac activity results 
 in more lateral protrusions and less directionally persistent migrational 
 behaviors , whereas lower rac activity inhibits lateral protrusions 
 and results in more directionally persistent migrational behaviors . it is possible that rac activity among the two different cell lines 
 is different . given that rac activity differs in 2d and 3d environments , 
 it will be interesting to determine if this difference in contact 
 guidance holds for cells migrating in 3d collagen networks . the finding 
 that different cell types interpret directional cues differently , 
 even though they bind and migrate in response to those cues , might 
 have a dramatic impact on cancer diagnosis . robust reorganization 
 and radial alignment of the collagen ecm around breast tumors has 
 led others to suggest that these could be used as diagnostic markers . we believe this to be a good approach , but perhaps aligned collagen 
 fibers are a necessary , but not sufficient signal that leads to cell 
 migration and cancer metastasis . we 
 demonstrated that the organization of collagen fibrils epitaxially 
 grown on mica surfaces induces a variety of morphological and migrational 
 behaviors depending on the specific organizational pattern of collagen . mda - mb-231 cells that migrate using a mesenchymal mode of migration 
 spread , elongated , oriented , and engaged in high fidelity contact 
 guidance on the highly aligned fibrils of large width and height . as fibril organization became more random , cell orientation and contact 
 guidance fidelity decreased . interestingly , mtln3 cells that migrate 
 using an amoeboid mode of migration spread and somewhat elongated 
 on aligned fibrils of large width and height , but did not engage in 
 contact guidance , even though they robustly migrated . this work demonstrates 
 that aligned collagen fibrils are a permissive , but not sufficient 
 signal for contact guidance and that the migrational mode of different 
 cells might impact whether a particular cell type senses directional 
 cues such as contact guidance .
invasion 
 of cancer cells into the surrounding tissue is an important 
 step during cancer progression and is driven by cell migration . 
 cell 
 migration can be random , but often it is directed by various cues 
 such as aligned fibers composed of extracellular matrix ( ecm ) , a process 
 called contact guidance . during contact guidance , aligned fibers bias 
 migration along the long axis of the fibers . 
 these aligned fibers 
 of ecm are commonly composed of type i collagen , an abundant structural 
 protein around tumors . in this paper 
 , we epitaxially grew several 
 different patterns of organized type i collagen on mica and compared 
 the morphology and contact guidance behavior of two invasive breast 
 cancer cell lines ( mda - mb-231 and mtln3 cells ) . 
 others have shown 
 that these cells randomly migrate in qualitatively different ways . 
 mda - mb-231 cells exert large traction forces , tightly adhere to the 
 ecm , and migrate with spindle - shaped morphology and thus adopt a mesenchymal 
 mode of migration . 
 mtln3 cells exert small traction forces , loosely 
 adhere to the ecm , and migrate with a more rounded morphology and 
 thus adopt an amoeboid mode of migration . 
 as the degree of alignment 
 of type i collagen fibrils increases , cells become more elongated 
 and engage in more directed contact guidance . 
 mda - mb-231 cells perceive 
 the directional signal of highly aligned type i collagen fibrils with 
 high fidelity , elongating to large extents and migrating directionally . 
 interestingly , behavior in mtln3 cells differs . 
 while highly aligned 
 type i collagen fibril patterns facilitate spreading and random migration 
 of mtln3 cells , they do not support elongation or directed migration . 
 thus , different contact guidance cues bias cell migration differently 
 and the fidelity of contact guidance is cell type dependent , suggesting 
 that ecm alignment is a permissive cue for contact guidance , but requires 
 a cell to have certain properties to interpret that cue .