The authors would like to specifically acknowledge Carolyn Kim, Amelia Lao, Ryan Stoutamore, and Itay Solomon for their contributions to earlier iterations of the photogelation scheme. Cell lines GS122 and GS304 were generously provided by David Nathanson. All figures were created with BioRender.com. UCLA core facilities, the Molecular Screening Shared Resources, and the Nano and Pico Characterization Laboratory were instrumental to the work. Chen Chia-Chun was supported by the UCLA Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research Training Program. Grigor Varuzhanyan was supported by a Tumor Cell Biology Training Program NIH Grant (T32 CA 009056).

Patient-derived GBM cell lines (GS122 and GS304) were provided by Professor David Nathanson (our collaborator), who developed these lines under a protocol approved by the UCLA Institutional Review Board (IRB# 10-000655). Cells were provided de-identified so that the cell lines could not be linked back to the individual patients.
1. Preparation of hydrogel solution
<ol>
	<li>Prepare HEPES-buffered solution by dissolving HEPES powder at 20 mM in Hank&#39;s balanced salt solution (HBSS). Adjust pH to 7 following full solvation.</li>
	<li>In the HEPES-buffered solution, dissolve thiolated HA (700 kDa nominal molecular weight, see Table of Materials), prepared following the previous report31, so that 6%-8% of carboxylic acid residues on each glucuronic acid are modified with a thiol, at a concentration of 10 mg/mL in buffer solution. 
	NOTE: An amber vial is recommended to prevent thiol oxidation by ambient light.
	<ol>
		<li>Stir using a magnetic stir plate (&#60;1,000 rpm) at room temperature until fully dissolved, typically around 45 min.</li>
	</ol>
	</li>
	<li>While HA is dissolving, prepare separate solutions of (1) 100 mg/mL of 8-arm-PEG-Norbornene (20 kDa), (2) 100 mg/mL of 4-arm-PEG-Thiol (20 kDa), (3) 4 mM of cysteine or cysteine-containing peptide (e.g., GCGYGRGDSPG), and (4) 4 mg/mL of LAP in microcentrifuge tubes (see Table of Materials).
	<ol>
		<li>Prepare each of these four solutions in the HEPES-buffered solution prepared in step 1.1. Vortex the solutions to ensure full dissolution of each reagent prior to performing step 4. 
		NOTE: If testing multiple different peptides, each must contain a cysteine or other source of thiol moiety for this conjugation chemistry.</li>
		<li>Prepare solutions (4 mM available thiol) of all peptides to be tethered within a single hydrogel at this point. 
		NOTE: Peptide sequences and ECM proteins from which they were derived and used in this study are listed in Table 1. N-acetyl cysteine (see Table of Materials), to which cells do not bind, can be substituted for a bioactive, thiol-containing peptide to titrate the concentration of an adhesive peptide or act as a negative control31.</li>
	</ol>
	</li>
	<li>Mix the individual solutions of HA, PEG-Norbornene, PEG-thiol, and cysteine/thiol-containing peptides (see Table of Materials) to achieve the final concentrations for the final hydrogel matrices listed in Table 2. Stir (&#60;1,000 rpm) on a magnetic stir plate for at least 30 min to mix fully. 
	&#8203;NOTE: HA solutions are highly viscous and best handled using a positive displacement pipette (see Table of Materials). If a positive displacement pipette is unavailable, viscous solutions can also be dispensed with a standard micropipette by slowly pipetting using wide-orifice tips.</li>
</ol>
2. Illumination and photocrosslinking of hydrogels via an LED array
CAUTION: Wear UV protective eyewear and cover the illumination field with UV-absorbing material.
NOTE: The LED array described in this protocol consists of six sets of eight LEDs placed in series, as illustrated by the provided circuit diagram (Figure 1A). Each set of LEDs can be independently powered, which allows for up to six different irradiances per run. Supplementary File 1 contains screenshots corresponding to the following directions for further guidance.
<ol>
	<li>Download the Illumination Device.zip file from the Supplementary Coding Files. This directory contains the following files: Arduino.zip (Supplementary Coding File 1), Drivers.zip (Supplementary Coding File 2), GUI.zip (Supplementary Coding File 3), and Holder.zip (Supplementary Coding File 4). 
	NOTE: 3D Print the top and bottom portions for holding the circuit board in place (see Supplementary Coding Files for details).</li>
	<li>Download and install the microcontroller software (see Table of Materials).</li>
	<li>Download and install the GUI software (see Table of Materials). Refer to Supplementary File 1 for software operating instructions.</li>
	<li>Open Processing and install the controlIP5 library via clicking on Sketch &#62; Import Library &#62; Add Library. Then, search for controlIP5 in libraries and click on Install. Perform this for the very first time.</li>
	<li>Power the illumination device (see Table of Materials) using the 36 Volt power supply and connect it to a PC using a micro-USB cable. 
	NOTE: Some devices will not install drivers automatically for various Arduino nano boards. One set of drivers is provided in the device zip file.</li>
	<li>Open the Arduino.ino file, located in the Adruino.zip folder, using Arduino IDE.</li>
	<li>Compile the Arduino.ino file by clicking on the Checkmark button. Upload the compiled code by clicking on the Arrow button.</li>
	<li>Open the GUI.pde file, located in the GUI.zip folder, using Processing.</li>
	<li>Click on Run in the processing program to launch the graphical user interface for controlling the illumination device.</li>
	<li>In the graphical user interface window, click on Intensity for the column containing hydrogel precursor solution to be crosslinked and input the desired intensity. Click on the Time box and input desired time. For the solution provided in Table 2, this will be 15 s. 
	NOTE: End-users need to calibrate digital intensity values to irradiance using a radiometer. Examples of typical intensities are provided in Figure 2A.</li>
	<li>Align the samples with the illumination device (Figure 2B) with every other LED in a single column of the silicone molds (see Table of Materials) or 384-well plate. Click on Finish to begin illumination. Repeat this process as necessary for illumination of multiple slides or other wells of a 384-well plate. 
	NOTE: The holder is designed such that the 384-well plate sits flush with one corner of the inner chamber during illumination.
	<ol>
		<li>Following illumination, when placed in one corner, move the well plate to the next corner and repeat. To illuminate wells on the other half of the plate, lift the plate out of the holder and rotate 180&#176;.</li>
	</ol>
	</li>
	<li>Generate hydrogels with varying mechanics for mechanical characterization following the steps below.
	<ol>
		<li>Clean the glass slides and silicone molds using tape to remove debris. Adhere the silicone molds to the glass slide, press down to ensure a good seal, and displace any air bubbles.</li>
		<li>Pipette 80 &#181;L of hydrogel precursor solution, as prepared in step 1.4, into each silicone mold on the glass slide.</li>
		<li>Place the glass slide onto the illumination device aligned with every other LED in a single column. Expose the hydrogel precursors to UV light for 15 s, as described in step 2, to photocrosslink.</li>
		<li>Once illumination has stopped, retrieve the slides, and loosen the gels from the molds by tracing the inner circumference of the mold with a fine tip (10 &#181;L pipette tip, 30 G needle, etc.). Remove silicone molds with tweezers/forceps.</li>
		<li>Move crosslinked hydrogels into individual wells of a 12-well plate by wetting a spatula and gently pushing them off the glass slide. Fill each well with 2 mL of DPBS (see Table of Materials) prior to adding the hydrogel. Swell the gels in DPBS solution for at least 12 h (typically overnight) at room temperature (for the next day&#39;s mechanical characterization).</li>
	</ol>
	</li>
</ol>
3. Atomic Force Microscopy (AFM) measurements
<ol>
	<li>Turn on the atomic force microscope (AFM) according to the manufacturer&#39;s instructions (see Table of Materials). This protocol provides brief instructions for using the instrument and the related software.</li>
	<li>Install the AFM probe (see Table of Materials). 
	NOTE: For the present study, a triangular silicon nitride cantilever with a nominal spring constant of 0.01 N/m was modified with a spherical 2.5 &#181;m silicon dioxide particle.</li>
	<li>Following installation, align the laser to the apex of the triangular probe, and then adjust mirror and laser deflection to maximize signal sum (typically between 1.5-2.2 Volts).</li>
	<li>Immerse the probe in DPBS and wait for up to 15 min to obtain thermal equilibrium. Click on the Calibration button and select Contact-Dependent calibration. Click on the Collect Thermal Tuning button, and following data collection, select the peak around 3 kHz for calibration. 
	NOTE: Slight adjustment of the mirror and laser deflectors may be necessary following immersion into a liquid due to refractive index changes.</li>
	<li>Approach the surface of a Petri dish (plastic) by setting the Approach Parameters to Constant Velocity, a target height of 7.5 &#181;m, and an approach speed of 15 &#181;m/s. Enable Baseline Measurement Per Run for Approach so that the approach runs continuously and does not stop early due to drift in the deflector.</li>
	<li>Upon approach, set acquisition parameters for force mapping to 4 nN turnarounds, 2 &#181;m indentation distance, 1 &#181;m/s velocity, and 0 s contact time. Press the Start button to begin collecting a force curve on the plastic surface (e.g., a well plate).</li>
	<li>Return to the calibration window and select the portion of the force curve corresponding to contact and indentation of the plastic. Accept the calculated sensitivity and stiffness values for the probe to complete calibration.</li>
	<li>Following calibration, raise the AFM probe and place the hydrogel sample for interrogation. Approach hydrogel following the settings provided in step 5. 
	NOTE: During the approach procedure toward the hydrogel surface, the unit may mistakenly trigger the approached state. To verify the actual approach, obtain a force curve as in step 4.6. Repeat the approach procedure if the resulting curve does not show contact and resulting indentation.</li>
	<li>When the surface approach is successful, switch to the Force Mapping mode and set acquisition parameters to a map of 8 x 8 size with 40 &#181;m length per axis. Obtain force maps in various regions to assess the uniformity of stiffness measurements.
	<ol>
		<li>Interpret force curves using the software program JPK SPM Data Processing through a Hertz/Sneddon model fit (Equations 1 and 2, see Table 3 for the definition of all the variables) with the spherical geometry selected32,33,34. 
		<img alt="Equation 1" src="/files/ftp_upload/63791/63791eq01.jpg" style="margin: auto;" />&#160; &#160;Equation 132 
		<img alt="Equation 2" src="/files/ftp_upload/63791/63791eq02.jpg" style="margin: auto;" />&#160; &#160;Equation 232</li>
	</ol>
	</li>
</ol>
4. Setting up and drug treatment of 3D, matrix-embedded cultures
<ol>
	<li>Prepare desired cells as a single cell solution. 
	NOTE: Different cell types may require different passaging methods. A typical protocol for passaging a suspension culture of GBM spheroids from a T-75 flask is reported in reference31.</li>
	<li>Collect GBM spheroids (roughly 150 &#181;m in diameter) from a T-75 flask suspension culture into a 15 mL conical tube. Rinse the culture flask with 5 mL of DPBS to remove any residual cells and media and add this volume to the conical tube.</li>
	<li>Centrifuge the conical tube containing cells at 200 x g for 5 min at room temperature. Following centrifugation, remove the supernatant with a 5 mL serological pipette, taking care not to disturb the cell pellet, and resuspend in 5 mL of DPBS.</li>
	<li>Centrifuge at 200 x g for 5 min at room temperature to wash cells. Aspirate the supernatant with a 5 mL serological pipette, taking care not to disturb the cell pellet, and then resuspend cells in 2 mL of cell dissociation reagent (see Table of Materials).</li>
	<li>Incubate at room temperature for 10-15 min. Add 3 mL of complete medium (see Table of Materials) and gently pipette 3-5 times to break down the spheroids to a single cell suspension31.</li>
	<li>Centrifuge the single-cell suspension at 400 x g (single-cell suspensions may be spun faster for pellet formation) for 5 min to pellet cells at room temperature. Aspirate the supernatant with a 5 mL serological pipette, taking care not to disturb the cell pellet. Resuspend cells in 1 mL of complete medium. 
	NOTE: If the cells remain in clumps, rather than as single cells in suspension, following passaging, cells can be passed through a 40 &#181;m cell strainer to achieve a single cell suspension.</li>
	<li>Remove a portion of the cells for counting using a hemocytometer. Dilute this portion two-fold with trypan blue, which permeates cells with compromised viability. Count only the live, colorless cells. Typically, a T-75 seeded at 800,000 cells per flask yields 2-3 million cells after a week in culture.</li>
	<li>Determine the number of cells necessary for encapsulation. Transfer a volume of media containing the total number of cells needed into a sterile 1.7 mL microcentrifuge tube. Spin down at 400 x g for 5 min at room temperature. 
	NOTE: For example, a minimum of 2.5 million cells resuspended in 1 mL of gel volume is needed to encapsulate cells at 2.5 million cells/mL. A gel volume of 1 mL allows users to dispense 100 gel drops, where each gel drop is of 10 &#181;L volume. Preparing an extra ~20% volume of cells suspended in hydrogel solution is recommended to account for loss during pipette transfer. Thus, one would prepare 3 million cells and 1.2 mL of hydrogel precursor solution in this example. A minimum density of 500 thousand cells/mL is recommended.</li>
	<li>Aspirate the supernatant with a micropipette, taking care not to disturb the cell pellet. Resuspend the cell pellet in the hydrogel precursor solution, as prepared in step 1.4, mixing well by pipetting up and down with a 1,000 &#181;L micropipette 4-5 times.</li>
	<li>Load the cells into a repeat pipettor (see Table of Materials) set to dispense 10 &#181;L. To avoid bubbles and uneven dispensing, prime the repeat pipettor by dispensing an additional 1-2 times into a waste container.</li>
	<li>In each well of a 384-well plate, dispense 10 &#181;L of cells suspended in hydrogel solution from the repeat pipettor. Using the LED array, illuminate each well containing cells (step 2) for 15 s with intensities (example results in Figure 2A utilized intensities of 1.14, 1.55, 2.15, 2.74 mW/cm2) to achieve the desired mechanical properties. 
	NOTE: It is suggested to start with five replicates per experimental condition and scale up or down depending on the desired throughput and variance of the endpoint assay.</li>
	<li>Add 40 &#181;L of complete media to each well containing the cells. Add 50 &#181;L of DPBS to non-experimental, dry wells surrounding the gels to minimize losses due to evaporation.</li>
	<li>For GBM cells, add 40 &#181;L of the media-containing drug (e.g., TMZ, see Table of Materials) to achieve the final desired concentration (10 &#181;M-100 &#181;M in dimethylsulfoxide (DMSO) or vehicle (DMSO), accordingly, starting 3 days after encapsulation.</li>
</ol>
5. CCK8 proliferation assay
<ol>
	<li>Add 10 &#181;L of CCK8 reagent (see Table of Materials) to each well containing the cells. 
	NOTE: If performing this assay for the first time, include negative control wells such as media only or cell-free hydrogel in media.</li>
	<li>Incubate for 1-4 h according to the manufacturer&#39;s instructions. 
	NOTE: This time may vary as a function of cell type and density, and thus incubation times need to be tested for each application so that absorbance values fall within a linear range, a requirement for applying Beer&#39;s Law35.</li>
	<li>Read absorbances at 450 nm for all wells following incubation.</li>
	<li>Calculate the average absorbance at 450 nm obtained in step 3 for the vehicle condition for each group. Divide each drug-treated well by the average of the vehicle control per group.</li>
	<li>Calculate confidence intervals by generating bootstrap distributions (N = 10,000) through the percentile method36. 
	NOTE: Generally, one may utilize 95% confidence intervals and interpret conditions whose confidence intervals do not cross over 1 to be significant and warrant further investigation. Setting confidence intervals to 95% is congruent with setting a significance cutoff of p = 0.05. For the data shown in the results, there is utility in distinguishing conditions that either promote or inhibit matrix-mediated drug resistance, requiring a two-side analysis.</li>
</ol>

<ol>
	<li>Scannell, J. W., Blanckley, A., Boldon, H., Warrington, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosing+the+decline+in+pharmaceutical+R&amp;D+efficiency.">Diagnosing the decline in pharmaceutical R&amp;D efficiency.</a> Nature Reviews Drug Discovery. 11 (3), 191-200 (2012).</li><li>Waring, M. 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=An+analysis+of+the+attrition+of+drug+candidates+from+four+major+pharmaceutical+companies.">An analysis of the attrition of drug candidates from four major pharmaceutical companies.</a> Nature Reviews Drug Discovery. 14 (7), 475-486 (2015).</li><li>Khozin, S., Liu, K., Jarow, J. P., Pazdur, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+do+oncology+drugs+fail+to+gain+US+regulatory+approval.">Why do oncology drugs fail to gain US regulatory approval.</a> Nature Reviews Drug Discovery. 14 (7), 450-451 (2015).</li><li>Booth, B., Ma, P., Glassman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncology's+trials.+Market+indicators.">Oncology's trials. Market indicators.</a> Nature Reviews Drug Discovery. 2 (8), 609-610 (2003).</li><li>Da Ros, M., 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=Glioblastoma+chemoresistance:+The+double+play+by+microenvironment+and+blood-brain+barrier.">Glioblastoma chemoresistance: The double play by microenvironment and blood-brain barrier.</a> International Journal of Molecular Sciences. 19 (10), 2879 (2018).</li><li>Broekman, M. 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=Multidimensional+communication+in+the+microenvirons+of+glioblastoma.">Multidimensional communication in the microenvirons of glioblastoma.</a> Nature Reviews Neurology. 14 (8), 482-495 (2018).</li><li>Grundy, T. 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=Differential+response+of+patient-derived+primary+glioblastoma+cells+to+environmental+stiffness.">Differential response of patient-derived primary glioblastoma cells to environmental stiffness.</a> Scientific Reports. 6 (1), 1-10 (2016).</li><li>Gomez-Roman, N., Stevenson, K., Gilmour, L., Hamilton, G., Chalmers, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+3D+human+glioblastoma+cell+culture+system+for+modeling+drug+and+radiation+responses.">A novel 3D human glioblastoma cell culture system for modeling drug and radiation responses.</a> Neuro-Oncology. 19 (2), 229-241 (2017).</li><li>Simoni, R. D., 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=Basement+membrane+complexes+with+biological+activity.">Basement membrane complexes with biological activity.</a> Biochemistry. 25 (2), 312-318 (2002).</li><li>Xiao, W., 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-mimetic+3D+culture+platforms+allow+investigation+of+cooperative+effects+of+extracellular+matrix+features+on+therapeutic+resistance+in+glioblastoma.">Brain-mimetic 3D culture platforms allow investigation of cooperative effects of extracellular matrix features on therapeutic resistance in glioblastoma.</a> Cancer Research. 78 (5), 1358-1370 (2018).</li><li>Aisenbrey, E. A., Murphy, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+alternatives+to+Matrigel.">Synthetic alternatives to Matrigel.</a> Nature Reviews Materials. 5 (7), 539-551 (2020).</li><li>Spinelli, 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=Molecular+subtypes+and+differentiation+programmes+of+glioma+stem+cells+as+determinants+of+extracellular+vesicle+profiles+and+endothelial+cell-stimulating+activities.">Molecular subtypes and differentiation programmes of glioma stem cells as determinants of extracellular vesicle profiles and endothelial cell-stimulating activities.</a> Journal of Extracellular Vesicles. 7 (1), 1490144 (2018).</li><li>Ostrom, Q. T., Cioffi, G., Waite, K., Kruchko, C., Barnholtz-Sloan, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CBTRUS+statistical+report:+Primary+brain+and+other+central+nervous+system+tumors+diagnosed+in+the+United+States+in+2014-2018.">CBTRUS statistical report: Primary brain and other central nervous system tumors diagnosed in the United States in 2014-2018.</a> Neuro-Oncology. 23, (2021).</li><li>Stupp, R., 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=Radiotherapy+plus+concomitant+and+adjuvant+temozolomide+for+glioblastoma.">Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma.</a> New England Journal of Medicine. 352 (10), 987-996 (2005).</li><li>Brennan, C. W., 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=The+somatic+genomic+landscape+of+glioblastoma.">The somatic genomic landscape of glioblastoma.</a> Cell. 155 (2), 462-477 (2013).</li><li>Tomczak, K., Czerwi&#324;ska, P., Wiznerowicz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cancer+Genome+Atlas+(TCGA):+An+immeasurable+source+of+knowledge.">The Cancer Genome Atlas (TCGA): An immeasurable source of knowledge.</a> Contemporary oncology. 19, 68-77 (2015).</li><li>Lee, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temozolomide+resistance+in+glioblastoma+multiforme.">Temozolomide resistance in glioblastoma multiforme.</a> Genes and Diseases. 3 (3), 198-210 (2016).</li><li>Joo, K. M., 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=Patient-specific+orthotopic+glioblastoma+xenograft+models+recapitulate+the+histopathology+and+biology+of+human+glioblastomas+in+situ.">Patient-specific orthotopic glioblastoma xenograft models recapitulate the histopathology and biology of human glioblastomas in situ.</a> Cell Reports. 3 (1), 260-273 (2013).</li><li>Levy, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+animal+as+models:+Ethical+considerations.">The use of animal as models: Ethical considerations.</a> International Journal of Stroke. 7 (5), 440-442 (2012).</li><li>Phon, B. W. S., Kamarudin, M. N. A., Bhuvanendran, S., Radhakrishnan, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transitioning+preclinical+glioblastoma+models+to+clinical+settings+with+biomarkers+identified+in+3D+cell-based+models:+A+systematic+scoping+review.">Transitioning preclinical glioblastoma models to clinical settings with biomarkers identified in 3D cell-based models: A systematic scoping review.</a> Biomedicine &amp; Pharmacotherapy. 145, 112396 (2022).</li><li>Xiao, W., 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=Bioengineered+scaffolds+for+3D+culture+demonstrate+extracellular+matrix-mediated+mechanisms+of+chemotherapy+resistance+in+glioblastoma.">Bioengineered scaffolds for 3D culture demonstrate extracellular matrix-mediated mechanisms of chemotherapy resistance in glioblastoma.</a> Matrix Biology. 85-86, 128-146 (2020).</li><li>Brancato, V., Oliveira, J. M., Correlo, V. M., Reis, R. L., Kundu, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Could+3D+models+of+cancer+enhance+drug+screening.">Could 3D models of cancer enhance drug screening.</a> Biomaterials. 232, 119744 (2020).</li><li>Xu, X., Farach-Carson, M. C., Jia, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+in+vitro+tumor+models+for+cancer+research+and+drug+evaluation.">Three-dimensional in vitro tumor models for cancer research and drug evaluation.</a> Biotechnology Advances. 32 (7), 1256-1268 (2014).</li><li>Xiao, W., Ehsanipour, A., Sohrabi, A., Seidlits, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronic-acid+based+hydrogels+for+3-dimensional+culture+of+patient-derived+Glioblastoma+Cells.">Hyaluronic-acid based hydrogels for 3-dimensional culture of patient-derived Glioblastoma Cells.</a> Journal of Visualized Experiments: JoVE. (138), e58176 (2018).</li><li>Preston, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digestion+products+of+the+PH20+hyaluronidase+inhibit+remyelination.">Digestion products of the PH20 hyaluronidase inhibit remyelination.</a> Annals of Neurology. 73 (2), 266-280 (2013).</li><li>Kim, Y., Kumar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD44-mediated+adhesion+to+hyaluronic+acid+contributes+to+mechanosensing+and+invasive+motility.">CD44-mediated adhesion to hyaluronic acid contributes to mechanosensing and invasive motility.</a> Molecular Cancer Research. 12 (10), 1416-1429 (2014).</li><li>Pibuel, M. A., Poodts, D., D&#237;az, M., Hajos, S. E., Lompard&#237;a, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+scrambled+story+between+hyaluronan+and+glioblastoma.">The scrambled story between hyaluronan and glioblastoma.</a> The Journal of Biological Chemistry. 296, 100549 (2021).</li><li>Xiao, W., Sohrabi, A., Seidlits, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+the+glioblastoma+microenvironment+into+engineered+experimental+models.">Integrating the glioblastoma microenvironment into engineered experimental models.</a> Future Science OA. 3 (3), (2017).</li><li>Trombetta-Lima, M., 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=Extracellular+matrix+proteome+remodeling+in+human+glioblastoma+and+medulloblastoma.">Extracellular matrix proteome remodeling in human glioblastoma and medulloblastoma.</a> Journal of Proteome Research. 20 (10), 4693-4707 (2021).</li><li>Schregel, 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=Characterization+of+glioblastoma+in+an+orthotopic+mouse+model+with+magnetic+resonance+elastography.">Characterization of glioblastoma in an orthotopic mouse model with magnetic resonance elastography.</a> NMR in Biomedicine. 31 (10), 3840 (2018).</li><li>Xiao, W., Ehsanipour, A., Sohrabi, A., Seidlits, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronic-acid+based+hydrogels+for+3-dimensional+culture+of+patient-derived+glioblastoma+cells.">Hyaluronic-acid based hydrogels for 3-dimensional culture of patient-derived glioblastoma cells.</a> Journal of Visualized Experiments: JoVE. (138), e58176 (2018).</li><li>Guz, N., Dokukin, M., Kalaparthi, V., Sokolov, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=If+cell+mechanics+can+be+described+by+elastic+modulus:+Study+of+different+models+and+probes+used+in+indentation+experiments.">If cell mechanics can be described by elastic modulus: Study of different models and probes used in indentation experiments.</a> Biophysical Journal. 107 (3), 564-575 (2014).</li><li>Sneddon, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relation+between+load+and+penetration+in+the+axisymmetric+boussinesq+problem+for+a+punch+of+arbitrary+profile.">The relation between load and penetration in the axisymmetric boussinesq problem for a punch of arbitrary profile.</a> International Journal of Engineering Science. 3 (1), 47-57 (1965).</li><li>Soofi, S. S., Last, J. A., Liliensiek, S. J., Nealey, P. F., Murphy, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+elastic+modulus+of+MatrigelTM+as+determined+by+atomic+force+microscopy.">The elastic modulus of MatrigelTM as determined by atomic force microscopy.</a> Journal of Structural Biology. 167 (3), 216-219 (2009).</li><li>Mayerh&#246;fer, T. G., Popp, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beer's+law+-+Why+absorbance+depends+(almost)+linearly+on+concentration.">Beer's law - Why absorbance depends (almost) linearly on concentration.</a> Chemphyschem: A European Journal of Chemical Physics and Physical Chemistry. 20 (4), 511-515 (2019).</li><li>Puth, M. T., Neuh&#228;user, M., Ruxton, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+variety+of+methods+for+calculating+confidence+intervals+by+bootstrapping.">On the variety of methods for calculating confidence intervals by bootstrapping.</a> Journal of Animal Ecology. 84 (4), 892-897 (2015).</li><li>Lavrentieva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gradient+hydrogels.">Gradient hydrogels.</a> Advances in Biochemical Engineering/Biotechnology. 178, 227-251 (2020).</li><li>Zhu, D., Trinh, P., Li, J., Grant, G. A., Yang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gradient+hydrogels+for+screening+stiffness+effects+on+patient-derived+glioblastoma+xenograft+cellfates+in+3D.">Gradient hydrogels for screening stiffness effects on patient-derived glioblastoma xenograft cellfates in 3D.</a> Journal of Biomedical Materials Research. Part A. 109 (6), 1027-1035 (2021).</li><li>da Hora, C. C., Schweiger, M. W., Wurdinger, T., Tannous, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+glioma+models:+From+patients+to+dish+to+animals.">Patient-derived glioma models: From patients to dish to animals.</a> Cells. 8 (10), 1177 (2019).</li><li>Li, W., 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=Characterization+and+transplantation+of+enteric+neural+crest+cells+from+human+induced+pluripotent+stem+cells.">Characterization and transplantation of enteric neural crest cells from human induced pluripotent stem cells.</a> Molecular Psychiatry. 23 (3), 499-508 (2018).</li><li>Scaringi, C., Minniti, G., Caporello, P., Enrici, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+inhibitor+cilengitide+for+the+treatment+of+glioblastoma:+A+brief+overview+of+current+clinical+results.">Integrin inhibitor cilengitide for the treatment of glioblastoma: A brief overview of current clinical results.</a> Anticancer Research. 32 (10), 4213-4224 (2012).</li></ol>

The authors have nothing to disclose.

The current work presents methods to generate 3D, miniaturized cultures within HA-based while simultaneously altering matrix stiffness and peptides available for integrin engagement. This technique enables the systematic study of how matrix parameters affect cellular phenotypes (e.g., the viability of cancer cells exposed to chemotherapy) with increased throughput. Previous approaches, including that presented herein, have tuned hydrogel stiffness by varying the percent total polymer in the final formulation, where stiff...

The expected cost of developing a new drug has steadily risen over the past decade, with over $1 billion in current estimates1. Part of this expense is the high failure rate of drugs entering clinical trials. Approximately 12% of drug candidates ultimately earn approval from the United States (US) Food &#38; Drug Administration (FDA) in 2019. Many drugs fail in Phase I due to unanticipated toxicity2, while others that pass safety trials may fail due to a lack of efficacy3. This attrition due to non-efficacy can partly be explained by the fact that cancer models used during drug development are notoriously non-predictive of clinical efficacy4.
Functional disparities between in vitro and in vivo models may be attributed to removing cancer cells from their native microenvironment, including non-tumor cells and the physical ECM5,6. Commonly, research groups use commercially available culture matrices, such as Matrigel (a proteinaceous basement membrane matrix derived from mouse sarcomas) to provide cultured tumor cells with a 3D matrix microenvironment. Compared to 2D culture, 3D culture in membrane matrix has improved the clinical relevance of in vitro results7,8. However, culture biomaterials from decellularized tissues, including the membrane matrix, typically exhibit batch-to-batch variability that may compromise reproducibility9. Furthermore, matrices derived from tumors with different tissue origins from those studied may not provide the appropriate physiological cues10. Finally, cancers with high degrees of intratumoral heterogeneity have microenvironmental features that vary on a submicron-size scale and which the membrane matrix cannot be tuned to recapitulate11.
Glioblastoma (GBM), a uniformly lethal brain tumor with a median survival time of approximately 15 months, is a cancer for which treatment development has been particularly difficult12,13. The current standard of care for GBM consists of primary tumor resection, followed by radiotherapy, and then chemotherapy using temozolomide (TMZ)14. Yet, more than half of clinical GBM tumors exhibit treatment resistance through various mechanisms15,16,17. Predicting the efficacy of a treatment regimen for an individual patient is extremely difficult. Standard preclinical models used to predict individual outcomes consist of patient-derived tumor cells xenografted orthotopically into immunocompromised mice. While patient-derived xenografts can recapitulate many aspects of clinical GBM tumors and are valuable for preclinical models18, they are inherently expensive, low throughput, time-consuming, and involve ethical concerns19. Cultures of patient-derived cells, on 2D plastic surfaces or as spheroids, mostly avoid these issues. While patient-derived cells preserve&#160;genetic aberrations, their cultures in 2D or as suspended spheroids have been largely poor representations of patient-derived xenografts in rodents and original patient tumors20. Previously, we, and others, have shown that GBM cells cultured in a 3D ECM that mimics the mechanical and biochemical properties of brain tissue can preserve drug resistance phenotypes10,21,22,23.
Interactions between hyaluronic acid (HA), a polysaccharide abundant in the brain ECM and overexpressed in GBM tumors, and its CD44 receptor modulate the acquisition of drug resistance in vitro21,24,25,26,27.&#160;For example, the inclusion of HA within soft, 3D cultures increased the ability of patient-derived GBM cells to acquire therapeutic resistance. This mechano-responsivity was dependent on HA binding to CD44 receptors on GBM cells21. Additionally, integrin binding to RGD-bearing peptides, incorporated into 3D culture matrices, amplified CD44-mediated chemoresistance in a stiffness-dependent manner21. Beyond HA, the expression of several ECM proteins, many containing RGD regions, vary between normal brain and GBM tumors28. For example, one study reported that 28 distinct ECM proteins were upregulated in GBM tumors29. Within this complex tumor matrix microenvironment, cancer cells integrate mechanical and biochemical cues to yield a particular resistance phenotype, which depends on relatively small differences (e.g., less than an order of magnitude) in Young&#39;s modulus or density of integrin-binding peptides28,29,30.
The present protocol characterizes how tumor cells interpret unique combinations of matrix cues and identify complex, patient-specific matrix microenvironments that promote treatment resistance (Figure 1A). A photochemical method for generating miniaturized, precisely tuned matrices for 3D culture provides a large, orthogonal variable space. A custom-built array of LEDs, run by a microcontroller, was incorporated to photocrosslink hydrogels within a 384-well plate format to increase automation and reproducibility. Exposure intensity was varied across well to alter micro-mechanical properties of resulting hydrogels, as assessed using atomic force microscopy (AFM). While this manuscript does not focus on constructing the illumination array itself, a circuit diagram (Figure 1B) and parts list (Table of Materials) are provided as aids for device reproduction.
This report demonstrates the rapid generation of an array of GBM cells cultured in unique, 3D microenvironments in which Young&#39;s modulus (four levels across a single order of magnitude) and integrin-binding peptide content (derived from four different ECM proteins) were varied orthogonally. The approach was then used to investigate the relative contributions of hydrogel mechanics and ECM-specific integrin engagement on the viability and proliferation of patient-derived GBM cells as they acquire resistance to temozolomide (TMZ) chemotherapy.

<table><tbody><tr><td>1.1 kOhm resistors, 6 W</td><td>Digikey</td><td>35601k1ft</td><td></td></tr><tr><td>1.7 mL microcentrifuge tube</td><td>Genesse Scientific</td><td>21-108</td><td></td></tr><tr><td>15 mL conical tube</td><td>Fisher Scientific</td><td>14-959-70C</td><td></td></tr><tr><td>365 nm LED</td><td>Digikey</td><td>ltpl-c034uvh365</td><td></td></tr><tr><td>384 well plate</td><td>Bio Greiner One</td><td>781090</td><td></td></tr><tr><td>40 &amp;micro;m cell strainer</td><td>MTC bio</td><td>C4040</td><td></td></tr><tr><td>4-Armed thiol terminated polyethlene glycol (20 kDa)</td><td>Laysan Bio</td><td>4arm-PEG-SH-20K-1g</td><td></td></tr><tr><td>6 NPN BJTs</td><td>Digikey</td><td>2n5550ta</td><td></td></tr><tr><td>80 Ohm resistors, 0.125 W</td><td>Digikey</td><td>erjj-6enf80r6v</td><td></td></tr><tr><td>8-Armed norbornene terminated polyethylene glycol (20 kDa)</td><td>Jenkem Technology</td><td>A7025-1</td><td></td></tr><tr><td>Accutase</td><td>Innovative Cell Technologies</td><td>AT104500</td><td>&amp;nbsp;cell dissociation&amp;nbsp; reagent</td></tr><tr><td>AFM Probes</td><td>Novascan</td><td></td><td>0.01 N/m Nominal spring constant, 2.5 &amp;micro;m SiO&lt;sub&gt;2&lt;/sub&gt; particle</td></tr><tr><td>Arduino IDE</td><td>Arduino</td><td>1.8.19</td><td></td></tr><tr><td>Arduino Nano</td><td>Makerfire</td><td>Mini Nano V3.0 ATmega328P Microcontroller Board</td><td></td></tr><tr><td>bFGF</td><td>Peprotech</td><td>100-18B</td><td>20 ng/mL</td></tr><tr><td>CCK8</td><td>Abcam</td><td>ab228554</td><td></td></tr><tr><td>Centrifuge</td><td>Thermoscientific</td><td>sorvall legend xtr</td><td></td></tr><tr><td>CP100ST</td><td>Gilson</td><td>F148415</td><td>Pipette tips for positive displacement pipette</td></tr><tr><td>Cubis Semi-Micro Balance</td><td>Sartorius</td><td>MSA225S100DI</td><td></td></tr><tr><td>DMEM - F12 (50-50)</td><td>Life Technologies</td><td>11330057</td><td>1x</td></tr><tr><td>DMSO</td><td>Fisher Scientific</td><td>BP231-100</td><td></td></tr><tr><td>DPBS Ca (-) Mg (-)</td><td>Genesse Scientific</td><td>25-508</td><td></td></tr><tr><td>EGF</td><td>Peprotech</td><td>AF100-15</td><td>50 ng/mL</td></tr><tr><td>Ethanol, Anhydrous</td><td>Fisher Scientific</td><td>A405P</td><td>Add DI water to dilute to 70%</td></tr><tr><td>Fisherbrand Class B Amber Glass threaded vials</td><td>Fisher Scientific</td><td>03-339-23C</td><td></td></tr><tr><td>Fisherbrand Weighing Paper</td><td>Fisher Scientific</td><td>09-898-12B</td><td></td></tr><tr><td>G21 Supplement</td><td>Gemini Bio</td><td>400-160</td><td>50x</td></tr><tr><td>Hanks Balanced Salt Solution</td><td>Thermo Fisher Scientific</td><td>14175095</td><td></td></tr><tr><td>HCl, ACS, 12M</td><td>Sigma Aldrich</td><td>S25838A</td><td>Add DI water to dilute to 1 M</td></tr><tr><td>Heparin sodium salt from porcine intestinal mucosa</td><td>Sigma Aldrich</td><td>H3149-100Ku</td><td>25 &amp;micro;g/mL</td></tr><tr><td>HEPES</td><td>Sigma Aldrich</td><td>H7006-100G</td><td></td></tr><tr><td>Hot Air Gun</td><td>Wagner</td><td>HT1000</td><td></td></tr><tr><td>Integrin-binding sialoprotein (IBSP) peptide</td><td>Genscript</td><td>Custom Order</td><td>GCGYGGGGNGEPRGDTYRAY</td></tr><tr><td>Lithium phenyl-2,4,6 trimethylbenzoylphosphinate (LAP) , &gt;95%</td><td>Sigma Aldrich</td><td>900889-1G</td><td></td></tr><tr><td>Magnetic stir plate</td><td>Thermo Scientific</td><td>SP194715</td><td></td></tr><tr><td>Microcentrifuge</td><td>Thermo Scientific</td><td>Sorvall legend micro 21R</td><td></td></tr><tr><td>Microman E single Channel Pipettor</td><td>Gilson</td><td>FD10004</td><td>Positive displacement pipette</td></tr><tr><td>Micropipette Tips</td><td>Various Manufacturs</td><td></td><td>Various sizes</td></tr><tr><td>mLine micropipette</td><td>Sartorious</td><td></td><td></td></tr><tr><td>N-acetyl Cysteine</td><td>Sigma Aldrich</td><td>A7250-10G</td><td></td></tr><tr><td>Nanowizard 4</td><td>Bruker</td><td></td><td>AFM microscope</td></tr><tr><td>NaOH</td><td>Fisher Scientific</td><td>ss255-1</td><td>Add DI water to dilute to 1 M</td></tr><tr><td>Normoicin</td><td>Invivogen</td><td>ant-nr-1</td><td>500x</td></tr><tr><td>Osteopontin Peptide</td><td>Genscript</td><td>Custom Order</td><td>GCGYGTVDVPDGRGDSLAYG</td></tr><tr><td>Pipet Aid</td><td>Drummond</td><td>4000102</td><td></td></tr><tr><td>Plain Microscope Slides</td><td>Globe Scientific</td><td>1301</td><td></td></tr><tr><td>Press-To-Seal silicone Isolator, 12-4.5mm diam x 2mm deep</td><td>Grace Bio Labs</td><td>664201-A</td><td>Cut so that 8 individual molds are made from a single sheet</td></tr><tr><td>Processing</td><td>Processing</td><td>3.5.4</td><td></td></tr><tr><td>Repeater M4</td><td>Eppendorf</td><td>4982000322</td><td></td></tr><tr><td>Repeater Pipette Tips</td><td>Sartorious</td><td>30089430</td><td>1 mL sizes</td></tr><tr><td>RGD Peptide</td><td>Genscript</td><td></td><td>GCGYGRGDSPG</td></tr><tr><td>Scoth Tape</td><td></td><td></td><td></td></tr><tr><td>Serological Pipettes</td><td>Genesse Scientific</td><td>12-102,12-104</td><td>5,10 mL Pipettes</td></tr><tr><td>Solder Paste</td><td>Digikey</td><td>315-NC191LT15T5-ND</td><td></td></tr><tr><td>Solder Wire</td><td></td><td></td><td></td></tr><tr><td>Straight dissecting forceps</td><td>VWR Scientific</td><td>82027-408</td><td></td></tr><tr><td>Synergy H1 Plate Reader</td><td>Biotek</td><td></td><td></td></tr><tr><td>T-75 Cell Culture Treated Flask</td><td>Genesee Scientific</td><td>25-209</td><td></td></tr><tr><td>Temozolomide</td><td>Sigma Aldrich</td><td>T2577</td><td>Typically used from 10 &amp;micro;M to 100 &amp;micro;M</td></tr><tr><td>Tenascin-C Peptide</td><td>Genscript</td><td></td><td>GCGYGRSTDLPGLKAATHYTITIR&lt;br/&gt; GV</td></tr><tr><td>Thiolated Hyaluronic Acid (700 kDa), 6-8% modified</td><td>Lifecore Biomedical</td><td>HA700K5</td><td></td></tr><tr><td>VWR Spinbar, Flea Micro</td><td>VWR</td><td>58948-375</td><td></td></tr></tbody></table>

hydrogel arrays enable increased throughput for screening effects of matrix components and therapeutics in 3d tumor models

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes and therapeutic responses. Accounting for matrix effects earlier in the drug development pipeline is expected to increase the likelihood of clinical success of novel therapeutics. Biomaterial-based strategies recapitulating specific tissue microenvironments in 3D cell culture exist but integrating these with the 2D culture methods primarily used for drug screening has been challenging. Thus, the protocol presented here details the development of methods for 3D culture within miniaturized biomaterial matrices in a multi-well plate format to facilitate integration with existing drug screening pipelines and conventional assays for cell viability. Since the matrix features critical for preserving clinically relevant phenotypes in cultured cells are expected to be highly tissue- and disease-specific, combinatorial screening of matrix parameters will be necessary to identify appropriate conditions for specific applications. The methods described here use a miniaturized culture format to assess cancer cell responses to orthogonal variation of matrix mechanics and ligand presentation. Specifically, this study demonstrates the use of this platform to investigate the effects of matrix parameters on the responses of patient-derived glioblastoma (GBM) cells to chemotherapy.

AFM measurements confirmed precise control of hydrogel mechanics as a function of UV irradiance (mW/cm2) during photo-crosslinking using a custom-built, Arduino-controlled LED array (Figure 2A). The hydrogel formulation used in this protocol can be found in Table 2. The spacing of the LEDs on the provided template matches the spacing for every other well of a 384-well plate, allowing for the formation of gels inside the plate (Figure 2B</stron...

Watch this Scientific Journal Video about Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models at JoVE.com

Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models

The present protocol describes an experimental platform to assess the effects of mechanical and biochemical cues on chemotherapeutic responses of patient-derived glioblastoma cells in 3D matrix-mimetic cultures using a custom-made UV illumination device facilitating high-throughput photocrosslinking of hydrogels with tunable mechanical features.

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes ...

hydrogel-arrays-enable-increased-throughput-for-screening-effects

Research

JoVE Journal

Bioengineering

2.5K Views.  University of California Los Angeles. Deze methode stelt onderzoekers in staat om systematisch te onderzoeken hoe matrix cues het fenotype van cellen in 3D-cultuur beïnvloeden. We demonstreren de procedure met behulp van culturen van GBM-cellen. Het belangrijkste voordeel van deze techniek is een implementatie met hoge doorvoer, waardoor we veel matrixcondities kunnen screenen.Deze techniek kan worden gebruikt om weefsel-mimetische matrices te ontwikkelen die specifieke functies in celkweekmodellen behouden, zoals medicij...

Video: Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models

Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array (HMCA)-based Plates

Cancer metastasis is known to cause 90% of cancer lethality. Metastasis is a multistage process which initiates with the penetration/invasion of tumor cells into neighboring tissue. Thus, invasion is a crucial step in metastasis, making the invasion process research and development of anti-metastatic drugs, highly significant. To address this demand, there is a need to develop 3D in vitro models which imitate the architecture of solid tumors and their microenvironment most closely to in vivo state on one hand, but at the same time be reproducible, robust and suitable for high yield and high content measurements. Currently, most invasion assays lean on sophisticated microfluidic technologies which are adequate for research but not for high volume drug screening. Other assays using plate-based devices with isolated individual spheroids in each well are material consuming and have low sample size per condition. The goal of the current protocol is to provide a simple and reproducible biomimetic 3D cell-based system for the analysis of invasion capacity in large populations of tumor spheroids. We developed a 3D model for invasion assay based on HMCA imaging plate for the research of tumor invasion and anti-metastatic drug discovery. This device enables the production of numerous uniform spheroids per well (high sample size per condition) surrounded by ECM components, while continuously and simultaneously observing and measuring the spheroids at single-element resolution for medium throughput screening of anti-metastatic drugs. This platform is presented here by the production of HeLa and MCF7 spheroids for exemplifying single cell and collective invasion. We compare the influence of the ECM component hyaluronic acid (HA) on the invasive capacity of collagen surrounding HeLa spheroids. Finally, we introduce Fisetin (invasion inhibitor) to HeLa spheroids and nitric oxide (NO) (invasion activator) to MCF7 spheroids. The results are analyzed by in-house software which enables semi-automatic, simple and fast analysis which facilitates multi-parameter examination.

Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array HMCA-based Plates

A HMCA-based imaging plate is presented for invasion assay performance. This plate facilitates the formation of three-dimensional (3D) tumor spheroids and the measurement of cancer cell invasion into the extracellular matrix (ECM). The invasion assay quantification is achieved by semi-automatic analysis.

Cancer metastasis is known to cause 90% of cancer lethality. Metastasis is a multistage process which initiates with the penetration/invasion of tumor ...

Cancer Research

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor microenvironment. To obtain more reliable in vitro results, several three-dimensional (3D) cell culture assays have been introduced. These assays allow cancer cells to interact with the extracellular matrix. This interaction affects cell behavior, such as proliferation and invasion, as well as cell morphology. Additionally, this interaction could induce or suppress the expression of several pro- and anti-tumorigenic molecules. Spheroid invasion assay was developed to provide a suitable 3D in vitro method to study cancer cell invasion. Currently, animal-derived matrices, such as mouse sarcoma-derived matrix (MSDM) and rat tail type I collagen, are mainly used in the spheroid invasion assays. Taking into consideration the differences between the human tumor microenvironment and animal-derived matrices, a human myoma-derived matrix (HMDM) was developed from benign uterus leiomyoma tissue. It has been shown that HMDM induces migration and invasion of carcinoma cells better than MSDM. This protocol provided a simple, reproducible, and reliable 3D human tumor-based spheroid invasion assay using the HMDM/fibrin matrix. It also includes detailed instructions on imaging and analysis. The spheroids grow in a U-shaped ultra-low attachment plate within the HMDM/fibrin matrix and invade through it. The invasion is daily imaged, measured, and analyzed using ilastik and Fiji ImageJ software. The assay platform was demonstrated using human laryngeal primary and metastatic squamous cell carcinoma cell lines. However, the protocol is suitable also for other solid cancer cell lines.

Tumor microenvironment is an essential part of cancer growth and invasion. To mimic carcinoma progression, a biologically relevant human matrix is needed. This protocol introduces an improvement for the in vitro three-dimensional spheroid invasion assay by applying a human leiomyoma-based matrix. The protocol also introduces a computer-based cell invasion analysis.

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor ...

Enhanced Viability for Ex vivo 3D Hydrogel Cultures of Patient-Derived Xenografts in a Perfused Microfluidic Platform

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically stable, thereby preserving molecular, genetic, and histological features, as well as heterogeneity of the original tumor. However, using these models to perform a multitude of experiments, including drug screening, is prohibitive both in terms of cost and time. Three-dimensional (3D) culture systems are widely viewed as platforms in which cancer cells retain their biological integrity through biochemical interactions, morphology, and architecture. Our team has extensive experience culturing PDX cells in vitro using 3D matrices composed of hyaluronic acid (HA). In order to separate mouse fibroblast stromal cells associated with PDXs, we use rotation culture, where stromal cells adhere to the surface of tissue culture-treated plates while dissociated PDX tumor cells float and self-associate into multicellular clusters. Also floating in the supernatant are single, often dead cells, which present a challenge in collecting viable PDX clusters for downstream encapsulation into hydrogels for 3D cell culture. In order to separate these single cells from live cell clusters, we have employed density step gradient centrifugation. The protocol described here allows for the depletion of non-viable single cells from the healthy population of cell clusters that will be used for further in vitro experimentation. In our studies, we incorporate the 3D cultures in microfluidic plates which allow for media perfusion during culture. After assessing the resultant cultures using a fluorescent image-based viability assay of purified versus non-purified cells, our results show that this additional separation step substantially reduced the number of non-viable cells from our cultures.

This protocol demonstrates methods to enable extended in vitro culture of patient-derived xenografts (PDX). One step enhances overall viability of multicellular cluster cultures in 3D hydrogels, through straightforward removal of non-viable single cells. A secondary step demonstrates best practices for PDX culture in a perfused microfluidic platform.

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically ...

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening.
Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. 
Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types.

We describe a set of protocols that together provide a tissue-mimicking hydrogel bioink with which functional and viable 3-D tissue constructs can be bioprinted for use in in vitro screening applications.

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell lines using conventional two-dimensional (2D) cell culture. These challenges can be overcome by using bioprinting techniques to build heterogeneous three-dimensional (3D) tumor models whereby different types of cells are embedded. Alginate and gelatin are two of the most common biomaterials employed in bioprinting due to their biocompatibility, biomimicry, and mechanical properties. By combining the two polymers, we achieved a bioprintable composite hydrogel with similarities to the microscopic architecture of a native tumor stroma. We studied the printability of the composite hydrogel via rheology and obtained the optimal printing window. Breast cancer cells and fibroblasts were embedded in the hydrogels and printed to form a 3D model mimicking the in vivo microenvironment. The bioprinted heterogeneous model achieves a high viability for long-term cell culture (&#62; 30 days) and promotes the self-assembly of breast cancer cells into multicellular tumor spheroids (MCTS). We observed the migration and interaction of the cancer-associated fibroblast cells (CAFs) with the MCTS in this model. By using bioprinted cell culture platforms as co-culture systems, it offers a unique tool to study the dependence of tumorigenesis on the stroma composition. This technique features a high-throughput, low cost, and high reproducibility, and it can also provide an alternative model to conventional cell monolayer cultures and animal tumor models to study cancer biology.

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

We developed a heterogeneous breast cancer model consisting of immortalized tumor and fibroblast cells embedded in a bioprintable alginate/gelatin bioink. The model recapitulates the in vivo tumor microenvironment and facilitates the formation of multicellular tumor spheroids, yielding insight into the mechanisms driving tumorigenesis.

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell ...

Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular matrix (ECM) of the unique brain environment, such as hyaluronic acid (HA), facilitates GBM progression and invasion. However, most in vitro culture models include GBM cells outside of the context of an ECM. Murine xenografts of GBM cells are used commonly as well. However, in vivo models make it difficult to isolate the contributions of individual features of the complex tumor microenvironment to tumor behavior. Here, we describe an HA hydrogel-based, three-dimensional (3D) culture platform that allows researchers to independently alter HA concentration and stiffness. High molecular weight HA and polyethylene glycol (PEG) comprise hydrogels, which are crosslinked via Michael-type addition in the presence of live cells. 3D hydrogel cultures of patient-derived GBM cells exhibit viability and proliferation rates as good as, or better than, when cultured as standard gliomaspheres. The hydrogel system also enables incorporation of ECM-mimetic peptides to isolate effects of specific cell-ECM interactions. Hydrogels are optically transparent so that live cells can be imaged in 3D culture. Finally, HA hydrogel cultures are compatible with standard techniques for molecular and cellular analyses, including PCR, Western blotting and cryosectioning followed by immunofluorescence staining.

Here, we present a protocol for three-dimensional culture of patient-derived glioblastoma cells within orthogonally tunable biomaterials designed to mimic the brain matrix. This approach provides an in vitro, experimental platform that maintains many characteristics of in vivo glioblastoma cells typically lost in culture.

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Medicine

Fabrication Followed by 3D Encapsulation of Tumor Spheroids in PEG Hydrogels

Tumor Spheroid Fabrication and Encapsulation in Polyethylene Glycol Hydrogels for Studying Spheroid-Matrix Interactions

Three-dimensional (3D) encapsulation of spheroids is crucial to adequately replicate the tumor microenvironment for optimal cell growth. Here, we designed an in vitro 3D glioblastoma model for spheroid encapsulation to mimic the tumor extracellular microenvironment. First, we formed square pyramidal microwell molds using polydimethylsiloxane. These microwell molds were then used to fabricate tumor spheroids with tightly controlled sizes from 50-500 &#956;m. Once spheroids were formed, they were harvested and encapsulated in polyethylene glycol (PEG)-based hydrogels. PEG hydrogels are a versatile platform for spheroid encapsulation, as hydrogel properties such as stiffness, degradability, and cell adhesiveness can be tuned independently. Here, we used a representative soft (~8 kPa) hydrogel to encapsulate glioblastoma spheroids. Finally, a method to stain and image spheroids was developed to obtain high-quality images via confocal microscopy. Due to the dense spheroid core and relatively sparse periphery, imaging can be difficult, but using a clearing solution and confocal optical sectioning helps alleviate these imaging difficulties. In summary, we show a method to fabricate uniform spheroids, encapsulate them in PEG hydrogels and perform confocal microscopy on the encapsulated spheroids to study spheroid growth and various cell-matrix interactions.

Author Spotlight: In Vitro Hydrogel Model for Glioblastoma Microenvironment Study

Here, we present a protocol that enables fast, robust, and cheap fabrication of tumor spheroids followed by hydrogel encapsulation. It is widely applicable as it does not require specialized equipment. It would be particularly useful for exploring spheroid-matrix interactions and building in vitro tissue physiology or pathology models.

Three-dimensional (3D) encapsulation of spheroids is crucial to adequately replicate the tumor microenvironment for optimal cell growth.  Here, we ...

Replicating Glioblastoma Invasion with Hyaluronan-Collagen I Hydrogel System

A Photopolymerizable Hyaluronic Acid-Collagen Model of the Invasive Glioma Microenvironment with Interstitial Flow

Glioblastoma recurrence is a major hindrance to treatment success and is driven by the invasion of glioma stem cells (GSCs) into healthy tissue that are inaccessible to surgical resection and are resistant to existing chemotherapies. Tissue-level fluid movement, or interstitial fluid flow (IFF), regulates GSC invasion in a manner dependent on the tumor microenvironment (TME), highlighting the need for model systems that incorporate both IFF and the TME. We present an accessible method for replicating the invasive TME in glioblastoma: a hyaluronan-collagen I hydrogel composed of human GSCs, astrocytes, and microglia seeded in a tissue culture insert. Elevated IFF can be represented by applying a fluid pressure head to the hydrogel. Additionally, this model can be tuned to replicate inter- or intra-patient differences in cellular ratios, flow rates, or matrix stiffnesses. Invasion can be quantified, while gels can be harvested for a variety of outcomes, including GSC invasion, flow cytometry, protein or RNA extraction, or imaging.

Author Spotlight: Patient-Informed 3D Model for Studying Glioblastoma Invasion via Interstitial Fluid Flow

We present a method for replicating the glioma tumor microenvironment at the invasive front that incorporates interstitial fluid flow. This model is a hyaluronan-collagen I hydrogel in a tissue culture insert where a fluid pressure head can be applied. Invasion can be quantified, and cells can be isolated or lysed.

Glioblastoma recurrence is a major hindrance to treatment success and is driven by the invasion of glioma stem cells (GSCs) into healthy tissue that are ...

Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models

Samenvatting

Meer video's verkennen