BIOPRINTING OF MICRODISSECTED TUMOR “CUBOIDS”

Introduction:: More than 96% of cancer drug candidates fail the FDA approval process [1]. One reason for this high attrition rate is that drug screening is performed on cell culture or animal models that lack the complexity of the human tumor microenvironment (TME) [2]. Functional drug tests on tumor biopsies which preserve the TME serve as higher-fidelity tests [3]. In our lab, we use regularly sized, cuboidal shaped, microdissected tumors (“cuboids”) because these tissues retain most of the TME [4]. To perform functional drug testing while still preserving the TME in a hydrogel environment, we bioprinted tumor cuboids in arrays of biodots consisting of cuboids in hydrogel. We used the Inkredible+ bioprinter. Bioprinting allows for a precise, controlled extrusion of biomaterials in their surrounding environment [5]. Bioink provides a matrix for live cells and tissues to support biological interactions. Additionally, cuboids are placed in a unique spatial position to interface with a microdevice. We show their viability in Cellink Hydrogel and their response to drug treatment.

Materials and Methods:: We performed 3 steps to bioprint cuboids (Fig. 1A). We mixed bioink with cuboids (500 cuboids/mL of bioink) and loaded the mix into a cartridge. We then printed two different arrays of Bioink dots. For one experiment, we printed a 3 by 3 array placed on top of a transwell insert inside a well plate (Fig.1B). We added CaCl₂ solution to the bottom of the transwell inserts to crosslink the bottom of the biodots for 10 minutes, maintaining their position. For the other experiment, we printed a 6 by 4 array on a microplate. To hold each biodot, we made circular traps (2 mm radius, 800 µm depth) with a laser cutter and immersed the dots with exposure to the CaCl₂ solution.

To print, we used Cellink, an insoluble cellulose-based hydrogel often used for 3D bioprinting. This ink allowed us to place cuboids in a specific position and make non-soluble biodots.

We prepared cuboids from mouse syngeneic breast cancer tumors (Py8119 and EO771-2) or from human glioma xenografts (U87) [4]. A RealTime-Glo viability assay was performed on Py8119 cuboids in media and in bioinks. Luminescence was measured by IVIS at day 1 (baseline) and at day 3. Biodots were treated with drugs (staurosporine, cisplatin and doxorubicin) for 3 days. DMSO was used as a vehicle control. After exposure to fluorescent dead nuclear stains, SYTOX green or Reddot2, we fixed and imaged the biodots.


Results, Conclusions, and Discussions:: We evaluated important printing parameters for biodots with microdissected cuboids. The biodot size varied with time extruded (Fig. 1C). Overall, dot volume and extrusion time had a positive linear correlation, though there was a stronger correlation with the less viscous Start Ink (Fig 1C). We chose 1.5 seconds as the extrusion time because it kept the volume of ink to a minimum of 4.09 (±.82) mL while extruding the maximum value of ~3.48 (±1.06) cuboids/dot. The number of cuboids per dot appeared to follow a Poisson distribution centered around the maximum value (~3.5 cuboids/dot) when using 500 cuboids/mL of bioink (Fig. 1C).

We performed a Realtime Glo assay to show the biocompatibility of cuboids in bioinks extruded with a syringe (Fig. 2A) Overall, the control conditions, Start Ink, Cellink and Crosslinked bioinks had similar luminescence ratios, thus showing similar viability (Fig. 2B)

We then showed drug responses of the cuboid biodots localized either on a transwell membrane or in a microplate (Fig. 3A, 3B, 4A). After exposure to fluorescent cell death dyes, cuboids from biodots on a transwell treated with cisplatin and staurosporine had higher fluorescence levels than the control, indicating cell death (Fig. 3C). Similarly, cuboids in biodots treated with Doxorubicin also had higher fluorescence levels than the DMSO vehicle control (Fig. 3D). On the microplate, cuboids in biodots treated with cisplatin or staurosporine also showed cell death (Fig. 4A, 4B, 4C).

These results validate our ability to perform drug treatments on intact tumor cuboids that have been bioprinted in Cellink bioink. They also validate our ability to use biodots arrays as a drug-treatment assay. Furthermore, our bioprinting approach can provide an automated way to localize and integrate cuboids in a microdevice such as a sensor or a gradient generator.


Acknowledgements (Optional): : This work was supported by NCI (R21CA251952 and 2R01CA181445).

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[3] R. K. Harrison, “Phase II and phase III failures: 2013-2015,” Nature Reviews Drug Discovery, vol. 15, no. 12, 2016, doi: 10.1038/nrd.2016.184.

[4] L. F. Horowitz et al., “Microdissected ‘cuboids’ for microfluidic drug testing of intact tissues,” Lab on a Chip, vol. 21, no. 1, 2021, doi: 10.1039/d0lc00801j.

[5] Dey M, Ozbolat IT. 3D bioprinting of cells, tissues and organs. Sci Rep. 2020 Aug 18;10(1):14023. doi: 10.1038/s41598-020-70086-y.