Hypoxic Jumbo Spheroids On-A-Chip (HOnAChip): Insights into Treatment Efficacy

Abstract

Hypoxia is a key characteristic of the tumor microenvironment, too rarely considered during drug development due to the lack of a user-friendly method to culture naturally hypoxic 3D tumor models. In this study, we used soft lithography to engineer a microfluidic platform allowing the culture of up to 240 naturally hypoxic tumor spheroids within an 80 mm by 82.5 mm chip. These jumbo spheroids on a chip are the largest to date (>750 µm), and express gold-standard hypoxic protein CAIX at their core only, a feature absent from smaller spheroids of the same cell lines. Using histopathology, we investigated response to combined radiotherapy (RT) and hypoxic prodrug Tirapazamine (TPZ) on our jumbo spheroids produced using two sarcoma cell lines (STS117 and SK-LMS-1). Our results demonstrate that TPZ preferentially targets the hypoxic core (STS117: p = 0.0009; SK-LMS-1: p = 0.0038), but the spheroids’ hypoxic core harbored as much DNA damage 24 h after irradiation as normoxic spheroid cells. These results validate our microfluidic device and jumbo spheroids as potent fundamental and pre-clinical tools for the study of hypoxia and its effects on treatment response.

Radiotherapy on-chip: microfluidics for translational radiation oncology

“…5C.iii). 54 Due to their high diameter, steep oxygen gradients are naturally formed in these spheroids, and gold-standard hypoxic proteins can be found in their core at depths similar to in vivo observations. The study screened 12 drug/RT combinations, using the hypoxic pro-drug tirapazamine (0, 10 μM and 35 μM) and γ-irradiation (0, 2, 4 and 8 Gy), and found both treatments to yield additive effects in either hypoxic or normoxic regions of the spheroids.…”

“…A few studies have conducted irradiation of microfluidic devices using cabinet irradiators, with biological samples, and will be explored more extensively in the following sections. 45,46,[53][54][55] More recently, FLASH-RT, a novel RT technique involving sources with extremely high dose-rates, has made its way into the preclinical setting. [56][57][58][59][60] Although the limited availability of FLASH-RT systems makes pre-clinical studies challenging, an appropriately designed microfluidic device compatible with this RT modality was recently tested by Grilj et al (Fig.…”

“…The study screened 12 drug/RT combinations, using the hypoxic pro-drug tirapazamine (0, 10 μM and 35 μM) and γ-irradiation (0, 2, 4 and 8 Gy), and found both treatments to yield additive effects in either hypoxic or normoxic regions of the spheroids. 54 Table 2 recapitulates the various sources, cancer types, combination therapies and endpoints used in these studies, as well as the different research avenues to which they belong.…”

“…Pre-clinical research Laboratory irradiators Cabinet irradiators (cell, small animals) 54,55,[62][63][64][65][66] γ (Cs-137), X-ray 1 660, 225 keV ∼0.03 Gy s −1 FLASH-RT 67 Intraoperative electron (Mobetron®, SIT) 68 e− 1 6 -9 MeV >100 Gy s −1 Proton beams (FLASH-PRT) 61,69 p+ 1.1 50-250 MeV >40 Gy s −1 External beam radiation therapy Conventional RT [3DCRT/IMRT/VMAT] Superficial/orthovoltage 46,47,70 X-ray 1 50-500 keV ∼0.03 Gy s −1 Teletherapy 71 Co-60 (γ) 1 1.25 MeV ∼0.05 Gy s −1 Linear accelerator (FFF)/TomoTherapy® 45,[72][73][74][75][76][77][78] X-ray, e−/X-ray 79 Gamma Knife® 75,80 Co-60 (γ) chip with high experimental predictability and precise evaluation of the delivered dose (Fig. 2B).…”

Cancer-on-a-Chip: Models for Studying Metastasis

“…Tumor cells actively migrate toward hypoxic vessels within the TME that act as a guiding cue. A jumbo spheroid-on-a-chip models has been introduced recently that can create a hypoxic 3D TME [69]. Conventionally, hypoxia is artificially induced by a hypoxic chamber, low O 2 incubator, chemicals, or other chemical inhibitors.…”