High-Throughput 3D Tumor Spheroid Array Platform for Evaluating Sensitivity of Proton-Drug Combinations

Proton beam therapy (PBT) is a critical treatment modality for head and neck squamous cell carcinoma (HNSCC). However, not much is known about drug combinations that may improve the efficacy of PBT. This study aimed to test the feasibility of a three-dimensional (3D) tumor-spheroid-based high-throughput screening platform that could assess cellular sensitivity against PBT. Spheroids of two HNSCC cell lines—Fadu and Cal27—cultured with a mixture of Matrigel were arrayed on a 384-pillar/well plate, followed by exposure to graded doses of protons or targeted drugs including olaparib at various concentrations. Calcein staining of HNSCC spheroids revealed a dose-dependent decrease in cell viability for proton irradiation or multiple targeted drugs, and provided quantitative data that discriminated the sensitivity between the two HNSCC cell lines. The combined effect of protons and olaparib was assessed by calculating the combination index from the survival rates of 4 × 4 matrices, showing that Cal27 spheroids had greater synergy with olaparib than Fadu spheroids. In contrast, adavosertib did not synergize with protons in both spheroids. Taken together, we demonstrated that the 3D pillar/well array platform was a useful tool that provided rapid, quantitative data for evaluating sensitivity to PBT and drug combinations. Our results further supported that administration of the combination of PBT and olaparib may be an effective treatment strategy for HNSCC patients.

Construction of a Fibroblast-Associated Tumor Spheroid Model Based on a Collagen Drop Array Chip

Spheroid, a 3D aggregate of tumor cells in a spherical shape, has overcome the limitations of conventional 3D cell models to accurately mimic the in-vivo environment of a human body. The spheroids are cultured with other primary cells and embedded in collagen drops using hang drop plates and low-attachment well plates to construct a spheroid–hydrogel model that better mimics the cell–cell and cell–extracellular matrix (ECM) interactions. However, the conventional methods of culturing and embedding spheroids into ECM have several shortcomings. The procedure of transferring a single spheroid at a time by manual pipetting results in well-to-well variation and even loss or damage of the spheroid. Based on the previously introduced droplet contact-based spheroid transfer technique, we present a poly(dimethylsiloxane) and resin-based drop array chip and a pillar array chip with alignment stoppers, which enhances the alignment between the chips for uniform placement of spheroids. This method allows the facile and stable transfer of the spheroid array and even eliminates the need for a stereomicroscope while handling the cell models. The novel platform demonstrates a homogeneous and time-efficient construction and diverse analysis of an array of fibroblast-associated glioblastoma multiforme spheroids that are embedded in collagen.

Mapping Tumor Spheroid Mechanics in Dependence of 3D Microenvironment Stiffness and Degradability by Brillouin Microscopy

Altered biophysical properties of cancer cells and of their microenvironment contribute to cancer progression. While the relationship between microenvironmental stiffness and cancer cell mechanical properties and responses has been previously studied using two-dimensional (2D) systems, much less is known about it in a physiologically more relevant 3D context and in particular for multicellular systems. To investigate the influence of microenvironment stiffness on tumor spheroid mechanics, we first generated MCF-7 tumor spheroids within matrix metalloproteinase (MMP)-degradable 3D polyethylene glycol (PEG)-heparin hydrogels, where spheroids showed reduced growth in stiffer hydrogels. We then quantitatively mapped the mechanical properties of tumor spheroids in situ using Brillouin microscopy. Maps acquired for tumor spheroids grown within stiff hydrogels showed elevated Brillouin frequency shifts (hence increased longitudinal elastic moduli) with increasing hydrogel stiffness. Maps furthermore revealed spatial variations of the mechanical properties across the spheroids’ cross-sections. When hydrogel degradability was blocked, comparable Brillouin frequency shifts of the MCF-7 spheroids were found in both compliant and stiff hydrogels, along with similar levels of growth-induced compressive stress. Under low compressive stress, single cells or free multicellular aggregates showed consistently lower Brillouin frequency shifts compared to spheroids growing within hydrogels. Thus, the spheroids’ mechanical properties were modulated by matrix stiffness and degradability as well as multicellularity, and also to the associated level of compressive stress felt by tumor spheroids. Spheroids generated from a panel of invasive breast, prostate and pancreatic cancer cell lines within degradable stiff hydrogels, showed higher Brillouin frequency shifts and less cell invasion compared to those in compliant hydrogels. Taken together, our findings contribute to a better understanding of the interplay between cancer cells and microenvironment mechanics and degradability, which is relevant to better understand cancer progression.

Correction: Sioud et al. Evaluation of In Vitro Phototoxicity of a Minibody-IR700 Conjugate Using Monolayer and Multicellular Tumor Spheroid Models. Cancers 2021, 13, 3356

The authors wish to make the following corrections to this paper [...]

191 GAPDH knock-in of high affinity CD16 in iPSC derived NK cells drives high-level expression and increased anti-tumor function

BackgroundNatural killer (NK) cells have emerged as an alternative cell type for clinical utility given the low propensity for graft-versus-host disease, thereby making NK cells a potential off-the-shelf cell therapy. One critical pathway NK cells use to target tumor cells is through expression of Fc gamma receptor III alpha (CD16). Antibodies that bind tumor antigens are recognized by CD16 on NK cells, promoting NK-mediated tumor cell killing. High-affinity CD16 variants in the human population correlate with better clinical outcome and anti-tumor response. One mechanism tumors use to evade NK cell recognition is through down-regulation of CD16 expression on the NK cell surface. After being activated, CD16 is cleaved by A Disintigrin and Metalloprotease-17 (ADAM-17). By using a highly-active engineered AsCas12a to knock-in high-affinity CD16 (hCD16KI) at the GAPDH locus, hCD16 is constitutively expressed, continuously replacing hCD16, thereby allowing for repeated ADCC mediated killing.Methods iPSCs were edited at the GAPDH locus with an engineered AsCas12a along with the CD16 donor construct. The bulk edited population was then plated at clonal density and single clones were selected and screened. iPSC clones were then differentiated into NK cells. A 3D tumor spheroid killing assay was used to demonstrate NK cell cytotoxicity against an ovarian cancer cell line (SKOV-3). In addition, a serial killing assay was used to better model NK cell serial killing.ResultsBi-allelic CD16KI iPSC clones were successfully generated. These iPSCs exhibited normal morphology and were able to differentiate into iNK cells. hCD16KI iNK cells showed normal differentiation and surface marker expression, such as CD45/CD56, compared to unedited iNK cells. CD16KI iNK cells demonstrated significantly increased cytotoxicity in the presence of antibody against tumor cells when compared with unedited iNK cells, as measured by reduction in tumor spheroid size in a 3D tumor spheroid killing assay. Importantly, enhanced surface expression of hCD16 on iNK cells after tumor exposure was detected, demonstrating the replenishment of cleaved hCD16. Notably, hCD16KI iNK cells demonstrated prolonged and enhanced tumor cell killing after being subjected to repeated tumor cell exposure in a serial killing assay.ConclusionsThis work demonstrates a powerful new method to drive high-level constitutive hCD16 expression on the surface of iNK cells through transgene knock-in at the GAPDH locus using an engineered AsCas12a. The high level constitutive hCD16 expression enhances ADCC of iNK cells and enables enhanced serial tumor killing and is expected to exert enhanced anti-tumor activity in the clinic.

Synergistic Cytotoxicity between Elephantopus scaber and Tamoxifen on MCF-7-Derived Multicellular Tumor Spheroid

Elephantopus scaber Linn, a traditional herb, exhibited anticancer properties, and it was cytotoxic against the monolayer estrogen receptor-positive breast cancer cell line, MCF-7, in the previous study. In order to determine the potential of E. scaber as a complementary medicine for breast cancer, this study aimed to evaluate the synergism between E. scaber and tamoxifen in cytotoxicity using MCF-7 in the form of 3-dimensional multicellular tumor spheroid (MCTS) cultures. MCTS represents a more reliable model for studying drug penetration as compared to monolayer cells due to its greater resemblance to solid tumor. Combination of E. scaber ethanol extract and tamoxifen, which were used in concentrations lower than their respective IC50 values, had successfully induced apoptosis on MCTS in this study. The combinatorial treatment showed >58% increase of lactate dehydrogenase release in cell media, cell cycle arrest at the S phase, and 1.3 fold increase in depolarization of mitochondrial membrane potential. The treated MCTS also experienced DNA fragmentation; this had been quantified by TUNEL-positive assay, which showed >64% increase in DNA damaged cells. Higher externalization of phospatidylserine and distorted and disintegrated spheroids stained by acridine orange/propidium iodide showed that the cell death was mainly due to apoptosis. Further exploration showed that the combinatorial treatment elevated caspases-8 and 9 activities involving both extrinsic and intrinsic pathways of apoptosis. The treatment also upregulated the expression of proapoptotic gene HSP 105 and downregulated the expression of prosurvival genes such as c-Jun, ICAM1, and VEGF. In conclusion, these results suggested that the coupling of E. scaber to low concentration of tamoxifen showed synergism in cytotoxicity and reducing drug resistance in estrogen receptor-positive breast cancer.

Mapping tumor spheroid mechanics in dependence of 3D microenvironment stiffness and degradability by Brillouin microscopy

Altered biophysical properties of cancer cells and of their microenvironment contribute to cancer progression. While the relationship between microenvironmental stiffness and cancer cell mechanical properties and responses has been previously studied using two-dimensional (2D) systems, much less is known about it in a physiologically more relevant 3D context and in particular for multicellular systems. To investigate the influence of microenvironment stiffness on tumor spheroid mechanics, we first generated MCF-7 tumor spheroids within matrix metalloproteinase (MMP)- degradable 3D polyethylene glycol (PEG)-heparin hydrogels, where spheroids showed reduced growth in stiffer hydrogels. We then quantitatively mapped the mechanical properties of tumor spheroids in situ using Brillouin microscopy. Maps acquired for tumor spheroids grown within stiff hydrogels showed elevated Brillouin frequency shifts (hence increased longitudinal elastic moduli) with increasing hydrogel stiffness. Maps furthermore revealed spatial variations of the mechanical properties across the spheroids cross-sections. When hydrogel degradability was blocked, comparable Brillouin frequency shifts of the MCF-7 spheroids were found in both compliant and stiff hydrogels, along with similar levels of growth-induced compressive stress. Under low compressive stress, single cells or free multicellular aggregates showed consistently lower Brillouin frequency shifts compared to spheroids growing within hydrogels. Thus, the spheroids mechanical properties were modulated by matrix stiffness and degradability as well as multicellularity, and also to the associated level of compressive stress felt by tumor spheroids. Spheroids generated from a panel of invasive breast, prostate and pancreatic cancer cell lines within degradable stiff hydrogels, showed higher Brillouin frequency shifts and less cell invasion compared to those in compliant hydrogels. Taken together, our findings contribute to a better understanding of the interplay between cancer cells and microenvironment mechanics and degradability, which is relevant to better understand cancer progression.