2 Tumor-induced remote ECM network orientation steers angiogenesis

Tumor angiogenesis promotes tumor growth and metastasis. Here, we use automated sequential microprinting of tumor and endothelial cells in extracellular matrix (ECM) scaﬀolds to study its mechanical aspects. Quantitative reﬂection microscopy shows that tumor spheroids induce radial orientation of the surrounding collagen ﬁber network up to a distance of ﬁve times their radius. Across a panel of ∼ 20 diﬀerent human tumor cell lines, remote collagen orientation is correlated with local tumor cell migration behavior. Tumor induced collagen orientation requires contractility but is remarkably resistant to depletion of collagen-binding integrins. Microvascular endothelial cells undergo directional migration towards tumor spheroids once they are within the tumor-oriented collagen ﬁber network. Laser ablation experiments indicate that an intact physical connection of the oriented network with the tumor spheroid is required for mechanical sensing by the endothelial cells. Together our ﬁndings show that remote physical manipulation of the ECM network by the tumor steers angiogenesis. our a remote mechanical cue to steer angiogenesis. remote tumor-mediated collagen network orientation. We show that tumor spheroids reorient a surrounding collagen-based ECM network up to ﬁve times their radius. In a panel of cell lines the distance of collagen orientation correlates with spheroid expansion which is mainly caused by tumor invasion/migration. Such long range collagen reorganization has also been observed for mouse ﬁbroblast explants

C h a p t e r 2 Tumor-induced remote ECM network orientation steers angiogenesis 1

Introduction
Tumor-associated angiogenesis is one of the hallmarks of cancer [1,2]. The chemotactic aspect of this pathology has been well studied. Oncogenic signaling pathways and hypoxia occurring in tumors activate the release of growth factors, such as vascular endothelial growth factor (VEGF), which triggers the formation of new microvascular sprouts from pre-existing vessels. Inhibitors against this paracrine interaction, targeting mainly the VEGF receptor (VEGFR) on endothelial cells, have entered the clinic [3,4].
Angiogenesis involves proliferation and migration of endothelial cells [5]. During angiogenesis, endothelial cells migrate through a 3D extracellular matrix (ECM) network that is rich in collagen. Migration efficiency and the mode of migration (e.g. the extent of integrin-dependency and the requirement of matrix metalloproteases) are determined by ECM properties including ligand density, stiffness, fiber crosslinking, and pore size [6][7][8]. These ECM properties are typically altered in tumor areas, e.g. ECM stiffening has been observed in tumor tissue [9,10].
The ECM network may control angiogenesis in several ways. First, it acts as an organizing platform for growth factor distribution, activation, and presentation [11]. In vitro assays have shown that tissue deformation can regulate angiogenesis through spatial organization of activity of the VEGF pathway [12]. In addition, cells receive mechanical cues from the ECM through integrin-based cell-matrix adhesions [13][14][15]. In vivo studies have demonstrated that angiogenesis is an integral response to chemical and mechanical cues [16].
Here we use sequential microprinting of tumor and microvascular endothelial cells to investigate their mechanical interaction through ECM scaffolds. We use quantitative reflection microscopy analysis to study tumor-induced collagen orientation. We show that tumor spheroids can orient a collagen network to a distance of up to 5 times the tumor radiusfar beyond the area of tumor expansion and cell migration. Furthermore, we demonstrate that microvascular endothelial cells sense and respond to such orientation provided that the oriented ECM is physically connected to the tumor spheroid. Together, our data indicates that ECM network reorientation acts as a remote mechanical cue to steer angiogenesis.

Results
2.2.1 Tumor spheroids in 3D collagen induce the reorientation of surrounding collagen 4T1 breast cancer spheroids were microprinted in collagen gels (Figure 2.1A) and their outgrowth and migration was monitored after 48 hours (Figure 2.1B). A spheroid mask was generated to define the final spheroid area including core spheroid and migrated cells (Figure 2.1C).
Reflection microscopy was performed to analyze the collagen network surrounding this final spheroid area (Figure 2.1D). In 2 days the tumor spheroid radius (including core and migrated cells) had increased ∼4-fold ( Figure 2.1A,B). Concomitantly, reflection microscopy showed that the surrounding collagen network contained an increase in radially oriented fibers (Figure 2.1D, 2.2A). Indeed, quantitative image analysis showed an increase in collagen fibers with an orientation parameter ∼1 (dark red; denoting collagen directed radially towards the tumor spheroid center) close to the spheroid boundary ( Figure 2.2A). By contrast, in areas distant from the spheroid, the number of fibers with an orientation parameter ∼1 equaled the number of fibers with an orientation parameter ∼0 (dark blue; denoting collagen oriented tangential to the tumor spheroid radius) (Figure 2.2A). Quantification of collagen fiber orientation throughout the gel relative to the distance from the final tumor spheroid edge (dashed red circle in Figure 2.1C,D) indicated that tumor spheroids that expanded from an average radius of 116±21 µm to 527±54 µm had caused radial orientation of collagen fibers up to 2.65 mm from the spheroid edge (i.e. 95% confidence interval >0.5 indicating orientation was significantly different from random) (Figure 2.2B). Thus, tumor spheroids induced remote orientation of collagen fibers up to distances of 5 times the spheroid radius.

Remote tumor-induced collagen network reorientation correlates with local cell migration capacity and requires Rho kinase-myosin activity
To address the role of collagen-binding integrins (mainly α1β1, α2β1) in tumor-induced collagen orientation we made use of cells stably expressing shRNAs targeting ITGB1, which express strongly reduced (∼90%) levels of β1 integrins [17]. For 4T1 cells, depletion of β1 integrins reduced  spheroid expansion through collective migration and induced migration of individual cells, as described before [17] (Figure 2.3A). This was accompanied by reduced collagen orientation (measured beyond the area of spheroid expansion -cell migration) (Figure 2.3B). Similar results were obtained for HCC70-derived tumor spheroids ( Figure S1A,B). By contrast, control MDA-MB-468 and BT20 tumor spheroids showed little migration whereas depletion of β1 integrins in these cells enhanced spheroid expansion through a mix of collective and single cell migration ( Figure 2.3C and S1C). In these cases, depletion of β1 integrins led to an increased remote collagen orientation ( Figure 2.3D and S1D). Lastly, in HCC1806 cells β1 integrin depletion caused a shift from relatively ineffective collective migration to similarly weak single cell migration and this did not affect the capacity of the tumor spheroids to cause collagen orientation ( Figure 2.3E,F). Together, these results indicated that the capacity of tumor cells to orient the collagen network was not affected by a reduction in collagen-binding integrins per se. Rather, changes in integrin expression caused decreased or increased tumor cell migration at the spheroid edge, which correlated with decreased or increased remote collagen orientation capacity, respectively.
We next tested a larger panel of carcinoma and sarcoma cell lines for their capacity to orient surrounding collagen ( Figure S2). In line with the results obtained with 4T1 cells, irrespective of the origin of the cell line, β1 integrin expression level, or migration strategy; there was a strong correlation between remote collagen orientation capacity and spheroid expansion (average initial spheroid radius for all cell lines was 113±29 µm) ( Figure 2.4A,B). Spheroid expansion as measured included spheroid growth and migration and tumor cell types showing the largest spheroid expansions typically displayed strong migration activity. To investigate the role of cytoskeletal contractility, pharmacological inhibition of myosin II or Rho kinase that acts upstream of myosin II activity was used for the duration of the experiment. Treatment of 4T1 spheroids with a myosin II inhibitor caused a ∼15% decrease in final spheroid radius and reduced collective migration activity that was accompanied by a 50% reduction in remote collagen orientation (Figure 2.4C,D and S3). Inhibition of Rho kinase led to a ∼8% decrease in final spheroid radius and caused a switch from collective migration to individual cell migration that was accompanied by a 70% reduction in remote collagen orientation ( Figure  2.4C,D and S3). These results showed that inhibition of Rho kinase-myosin II-mediated contractility has moderate effects on cell migration at the spheroid edge but strongly attenuates remote collagen orientation.

Endothelial response to oriented collagen network requires physical coupling with tumor
To address if physical connections between the tumor spheroid and the oriented collagen network remained important for guidance of endothelial cells, the spheroid was physically disconnected after the collagen network had been oriented. For this purpose, two HMEC-1 spheroids were injected at the same distance from the tumor spheroid at opposite sides and laser cutting of collagen fibers was applied close to the tumor edge, between the tumor and one of the HMEC-1 spheroids (Figure 2.7A). Orientation of the collagen network was maintained in areas disconnected from the tumor spheroid through laser ablation (Figure 2.7B,C). However, HMEC-1 cells injected in such areas no longer responded to collagen orientation: HMEC-1 spheroid direction, elongation, and the combined orientation parameter were decreased; resembling HMEC-1 behavior in non-oriented collagen areas (Figure 2.7D-F). Control HMEC-1 spheroids in the same well that were still connected to the tumor normally responded to oriented collagen. These findings demonstrate that an intact physical connection of the oriented ECM network with the tumor spheroid is required for orientation sensing by the endothelial cells.
Here, we use quantitative reflection microscopy analysis to study remote tumor-mediated collagen network orientation. We show that tumor spheroids reorient a surrounding collagen-based ECM network up to five times their radius. In a panel of cell lines the distance of collagen orientation correlates with spheroid expansion which is mainly caused by tumor invasion/migration. Such long range collagen reorganization has also been observed for mouse fibroblast explants [33]. Local ECM reorga-

nization in areas containing tumor cells is driven by Rho kinase-Myosin
II-mediated contractility [20,34,35]. Our findings indicate that traction forces applied by the tumor cells on the local collagen network drives ECM reorientation also in distant areas where tumor cells are absent. In fact, while consequences of contractility inhibition for local tumor cell migration are limited, which can be explained by tumor cell plasticity, remote ECM reorientation is strongly attenuated.
Antibody blocking experiments have shown that collagen-binding integrins mediate i) local tumor-induced collagen network reorganization [36], and ii) tumor cell-responses to mechanical ECM properties [25]. Gene silencing as used in our study may be less efficient than antibody blocking. Nevertheless, we observe highly distinct effects of β1 integrin silencing on collagen network reorientation. We and others have previously shown that depletion or blockade of β1 integrins can either inhibit migration or cause a switch from collective to single cell migration, e.g. through effects on TGF-β signaling [17,37,38]. Our current study shows that in tumors where collective migration is attenuated or switched to less abundant individual cell migration in response to β1 integrin silencing, collagen network reorientation is lost (e.g. 4T1); whereas in tumors where cell motility is normally very poor and β1 integrin silencing triggers more abundant (individual) cell migration, a concomitant increase in collagen network reorientation is observed (e.g. MDA-MB-468). The fact that β1 integrin silencing does not directly attenuate collagen organization may point to roles for other collagen-binding receptors. On stromal fibroblasts, syndecan-1 participates in ECM network alignment [39]. Likewise, syndecans or discoidin domain collagen receptors on tumor cells may be candidates for force-induced collagen reorganization in the context of strongly reduced integrin levels.
The experiments discussed above show that tumor spheroids can reorient the collagen network at relatively long distances, way beyond the area of tumor expansion and migration. We subsequently show that endothelial cells can sense such long-range orientation and respond by moving towards the tumor. It is known that mechanical ECM properties, such as density and stiffness regulate angiogenesis [40][41][42][43]. This may be explained by changes in the distribution of soluble factors or enhanced activity of the receptors for these factors [12,44,45]. Alternatively, physical aspects of the network may instruct endothelial cell behavior. Indeed, we show that tumor-mediated remote radial organi-zation of collagen directs human microvascular endothelial cells. The correlation between levels of remote collagen organization and induction of endothelial cell directionality holds through for a panel of ∼20 different human cancer cell lines. Importantly, laser ablation of collagen fibers close to the tumor does not affect the architecture of the remote collagen network but leads to complete loss of endothelial cell responsiveness to such oriented ECM regions. This argues against a mechanism involving chemotactic signals. It also indicates that contact guidance, i.e. a preference for aligned collagen fibers, is insufficient. Rather, once the collagen network is organized, distant forces applied to the network by the tumor are critical for sensing and/or responding of endothelial cells.
Taken together, our study shows for the first time that a radial collagen network organization generated by the tumor relatively far beyond the area of tumor expansion and migration, not only forms migratory highways for tumor invasion but can also guide angiogenesis in a manner dependent on tumor generated traction forces. In coordination with soluble factors, this mechanical interaction might further direct microvascular sprouts towards the tumor. Hence, targeting tumor induced ECM remodeling may prevent both tumor invasion and angiogenesis.

Automated sequential microprinting of tumor-and endothelial cells in ECM scaffolds
Collagen type I solution was isolated from rat-tail collagen by acid extraction as described previously [49]. Collagen was diluted to 1 mg/mL in the culture medium containing 0.1 M Hepes (BioSolve) and fixed to pH 7.5 by addition of NaHCO 3 (stock 440 mM, Merck). 60 µL of this solution was then pipetted into a glass-bottom 96 well plate (Greiner) and incubated for 1 hour at 37°C to polymerize. Automated injection of cell suspensions into the resulting collagen gels to generate arrays of cell spheroids with defined x-y-z position was performed as described using injection robotics from Life Science Methods, Leiden NL (http://www.lifesciencemethods.com) [17,50]. Tumor spheroids of 113±29 µm initial radius were generated at 200 µm above the glass surface (average collagen gel height ∼1.5 mm) and incubated 48 hours with appropriate culture media for each cell line. Subsequently, medium was removed, HMEC-1 cells were injected at the same z-position at various defined x-y distances from the tumor spheroid, and wells were further incubated with HMEC-1 culture media for 24 hours ( Figure S4).
For experiments where tumor spheroids were treated with Myosin II or Rho kinase inhibitors, media was supplemented with blebbistatin (Calbiochem cat. number 203389, Merck KGaA, Darmstadt, Germany) or Y27632 (Tocris cat. number 1254, Bristol, UK), respectively reaching 10 µM final concentration (medium+gel). For fluorescent imaging of 4T1 and HMEC-1, cells were incubated at 37°C with 1 µM CellTracker Orange CMRA or CellTracker Green CMFDA Dye, respectively for 15 minutes prior to injection.

Collagen gel imaging
Spheroids were imaged using a Nikon TE2000 confocal microscope equipped with a Prior stage controlled by NIS Element Software and with a temperature and CO 2 -controlled incubator. Frame stitching was used when necessary. Differential interference contrast (DIC) images were captured using a charged coupled device (CCD) camera with NIS software and 10x dry objective. Reflection microscopy of the entire well was performed by 5.4 mm x 5.4 mm stitching of images obtained using a 40x long distance water immersion objective by illuminating with a 561 nm laser coupled with a 561 nm blocking dichroic mirror for the detection.

Laser severing assay
After injecting HMEC-1 cells on both sides of the tumor spheroid at a distance of 1650 µm from the tumor spheroid center, laser severing was performed by applying 16 lines/second stimulation just outside of the tumor spheroid with infrared laser (Coherent Chameleon Discovery) at 790 nm wavelength at full power (∼3000 mW), using the 40x long distance water immersion lens, while manually scanning through the z plane over a duration of five minutes. This was repeated until all the collagen at one side of the tumor spheroid was cut.

Image analysis
All image analysis was performed using in house written Matlab scripts (Mathworks, Natick, MA, USA). DIC images were first put through a median filter to create a background illumination signal to which the original images were normalized. Normalized images were blurred and a mask for core detection was generated by thresholding for signal lower than two standard deviations below the mean and taking only the central binary image. Subsequently, a canny edge detection method was applied to the normalized image to mask the outer rim of the spheroid. This mask was dilated to include the area of single cell migration and combined with the core mask to capture the entire final spheroid.
For reflection image analysis, first a background image was calculated by applying a circular averaging filter of 10 pixel radius to the original image. This image was then subtracted from the original image and a customized rollingball filter was applied to extract fibrillar structures. The filter multiplied the signal with itself and used a local thresholding algorithm assigning pixels with squared intensities >0.5 standard deviations above mean squared intensity within 5px distance, to a collagen fiber. From this binary image, isolated pixels were removed, a binary closure was performed, and structures of >20 pixels and eccentricity >0.9 were assigned as fibers.
The directionality of a fiber was quantified by first manually determining the center of the tumor spheroid per image, and subsequently calculating for each fiber; the cosine square of the angle between the vector pointing from the tumor spheroid to the center of the collagen fiber and the orientation of the collagen fiber. The distance of a fiber to the tumor edge was calculated by subtracting the previously determined tumor spheroid radius (obtained from the DIC image analysis) from the distance of the fiber center to the tumor spheroid center. Fiber orientations were analyzed depending on their distance, in bins of 100px (67 µm). To this data a two-parameter single exponential plateauing at 0.5 was fitted with the equation Y=(p1-0.5)exp(-X/p2)+0.5 for x (distance) larger than 100 µm using GraphPad Prism 6 program (GraphPad Software, La Jolla, CA). Integrated orientation was calculated from the fit by taking the integral ∞ 0 (p1 − 0.5)exp(−X/p2) dx which yielded the result (p1-0.5)*p2.
The collagen organization at the locations of HMEC-1 spheroids that were injected at designated distances from tumor center was determined by quantifying the collagen organization at that distance from the tumor spheroid before the HMEC-1 injections were performed, except when quantifying collagen orientation for the laser severing experiment for which the collagen organization was quantified both before HMEC-1 injection and after HMEC-1 injection/laser severing was performed. The HMEC-1 direction was determined by calculating the angle between the vector pointing from the HMEC-1 center to the tumor spheroid center and HMEC-1 long axis obtained from the injection mask, subtracting this angle from 90 degrees and dividing by 90 degrees so that HMEC-1 directed towards the tumor had a direction of 1 and directed perpendicularly had a direction 0. The elongation was calculated by dividing the long axis by the short axis length for the injection mask. Pearson product-moment correlation coefficient and linear fit were obtained using GraphPad Prism 6 software. The spheroid orientation was calculated by multiplying the direction with the elongation parameter.
To calculate significance between two conditions, the Mann-Whitney U test was used when comparing distribution data, and unpaired t-test was used when comparing integrated collagen orientation.

Figure S1
Effects of β1 integrin downregulation on tumor spheroid cell migration and collagen orientation. (A,C,E) Collagen orientation images merged with brightfield images taken 48 hours after injecting the indicated cell lines with or without shRNA targeting ITGB1 (A,C) or without injection (E). (B,D,F) Collagen orientation measured at a range of distances from tumor border for HCC 70 shctrl (B, green; n=16), HCC 70 shITGB1 (B, red; n=15), BT20 shctrl (D, green; n=17), BT20 shITGB1 (D, red; n=17) tumor spheroids 48 hours after injection, and from the average injection location for empty well (F, black; n=22) at the same time point, mean ± standard deviation with exponential fits (solid lines) from at least three independent experimental replicas is shown. Scale bar, 200 µm.   (D) Collagen orientation measured at a range of distances from spheroid border for 4T1 injections after 48 hours without treatment (black, n=55), with 10 µM Y27632 (green, n=46) or 10 µM blebbistatin treatment (red, n=53) or from the average injection location for empty well (blue, n=23) at the same time point, mean ± standard deviation with exponential fits (solid lines) from three independent experimental replicas is shown. Scale bar, 200 µm.

Figure S4
Automated sequential microinjection layout for tumor spheroid-HMEC-1 interaction. Low magnification image of multiwell plate showing 4T1 cells (red arrow heads) injected at identical x-y-z position in each well followed by HMEC-1 cells (blue arrow heads) injected at varying distances, 48 hours later and incubated for an additional 24 hours. Scale bar, 3mm.