In-vitro assays for immuno-oncology drug efficacy assessment and screening for personalized cancer therapy: scopes and challenges

Figures & data

Table 1. Comparison of different 3D structural models for cancer research [30, 42, 65, 79, 80].

Type	Description	Advantages	Disadvantages	Duration to reach optimal size	Image
MCTS	Compact cellular aggregates forming a spherical shape	Easiest to generate, easy maintenance Comparatively cheap, High throughput Require minimal technical handling/modification Heterogenous Highly reproducible for several cancer cell types Preserve stromal and nonmalignant cellular component	Not suitable for long-time culture Require specially designed plates for size uniformity Large spheroids contain necrotic cores Suitable for a limited number of cell types Special media additives and pre-processing are required for some cell types	1–7 days (higher for patient-derived cells)	MCF-7 MCTS, image adapted from, ‘Generation of Multicellular Tumor Spheroids with Microwell-Based Agarose Scaffolds for Drug Testing’ by Gong X, Lin C, Cheng J, et al., 2015 [33].
Tumorosphere	Spheroids emerge from the proliferation of isolated single cancer cells (generally cancer stem cells)	Quickly reach optimal size A small number of cells are required even for mass production Can be used for a wide range of cancer cell types Moderately reproducible Cancer stemness preserved More uniform in size and shape	Mostly homogenous Cannot mimic TME Skilled technical handling and pre-processing required Difficult maintenance Comparatively costly Does not preserve the original characteristics of a tumor	5–30 days	Tumoro-sphere from clonal expansion of single neural stem cell, image adapted from, ‘Direct isolation of human central nervous system stem cells. Proceedings of the National Academy of Sciences’ by Uchida N et al., 2000. Copyright © 2000, The National Academy of Sciences [34].
TDTS	Spheroids are obtained by culturing minutely minced, crushed, and/or partially dissociated (enzymatic) tumor tissue	Easy and quick to grow Tumor heterogeneity is preserved Moderately high-throughput	Suitable for only a limited number of cancer types Slow growth rate Prone to hypoxia Stromal components can be lost	1–3 days	TDTS derived from colorectal tumor tissue, image adapted from, ‘Retaining cell-cell contact enables preparation and culture of spheroids composed of pure primary cancer cells from colorectal cancer’ by Kondo J et al., 2011 [35].
OMS	Tumor tissue is sliced into minute pieces and cultured in media without enzymatic digestion	Characteristically closest to original in-vivo tumor Preserve tumor heterogeneity Stromal components are preserved Easy and quick to grow	Suitable for only a limited number of cancer types Cancer stemness cannot be studied	2–5 days until 12–18 days	OMS from bladder tumor sample, image adapted from, ‘A Chemosensitivity Test for Superficial Bladder Cancer Based on Three-Dimensional Culture of Tumour Spheroids’ by Burgués JP et al., 2007 [36].
PDE	Minutely sliced tissue fragments are cultured either submerged in media or air-liquid interface. They are similar to OMS but the fragments are larger and spherical spheroids are not isolated	Closest to original in-vivo tumor Preserves tumor heterogeneity Stromal components are preserved Easy and quick to grow	Prone to hypoxia Cannot be cultured for a long time Application is limited Low throughput Requires large tissue sample Precision handling is required to get uniform micro fragments	0–1 day	Explant culture using NSCLC tumor fragments, image adapted from, ‘Patient-derived explants (PDEs) as a powerful preclinical platform for anti-cancer drug and biomarker discovery’ by Powley IR et al., 2020 [37].
3D bioprinting	Specialized devices use cell suspension as bio-ink to seed cells of interest in specific patterns to form 3D shapes	Can be used for a wide range of cells Automated handling can reduce contamination 3D structures can be created of different shapes Customized 3D shapes can be formed for specialized use High throughput	Costly High maintenance Specialized training required Still under development	3–30 days (depends on cell type, shape, and use)	Bioprinted chimeric organoid using MCF-12A and MDA-MB-468 cells, image adapted from, ‘3D bioprinting for reconstituting the cancer microenvironment’ by Datta P et al., 2020 [38].
Tumor-on-a-chip	Specialized microfluidic devices use cells to form spheroids in a perfusion-controlled environment	Different cell populations can be used to create complex microenvironment Both scaffold-free and scaffold-based conditions can be applied Easy to control environment (TME, flow, and other forces) Easy in-situ monitoring	Costly Long-term culture not reported Specialized training required Still under development Reproducibility, consistency, and high-throughput capability are not yet established	1–13 days	Patient-derived Lung adenocarcinoma spheroid cultured on a microfluidic chip, image adapted from, ‘Towards personalized medicine: chemosensitivity assays of patient lung cancer cell spheroids in a perfused microfluidic platform’ by Ruppen J et al., 2015 [39].

MCTS: Multicellular Tumor Spheroid, SCS: Single Cell Proliferation Spheroid, TDTS: Tissue Derived Tumor Spheroids, Organotypic Multicellular Spheroid, PDE: Patient-Derived Explant, TME: Tumor Microenvironment.

Table 2. Comparison of spheroid forming techniques [38,40–49,52,62,69,95–100].

Techniques	Description	Ease of use	Ease of maintenance	Cost effective-ess	Uniformity	Easy to harvest	High throughput	Quick duration	Long-term culture suitability	Automation capable	Cancer cell type coverage	TME preservation	Special consideration	Immunotherapy efficacy assessment example
Hanging Drop	Cell suspension drops are hung from an inverted glass/plastic surface, thus allowing cell aggregation using gravity	+++	+	+++	±	+++	-	+++	-	+	+	±	Careful handling is required	[40]
Suspension culture	Cells spontaneously aggregate to form spheroids on low-attachment plates	+++	+++	+++	-	+++	+++	++	-	+	+	±	Plate surface modification may be required	[41]
High viscosity suspension	Cells spontaneously aggregate to form spheroids on low-attachment plates/hanging drop settings, media additives are added to increase viscosity (i.e. methylcellulose) and facilitate cell aggregation	++	+	+++	-	++	+++	++	-	+	+	±	Plate surface modification may be required	[42]
Pellet Culture	Using a centrifuge, cells are concentrated as a pellet at the bottom of a small microcentrifuge tube and cultured for 1–2 days before transferring into ultra-low attachment plates for subsequent use	+++	++	+++	±	+++	+++	+++	-	±	++	+	Larger larger-sized spheroids, Shear force from centrifugation may damage cells	-
Spinner Flask	Specially designed spinning flasks containing single-cell suspension continuously agitate by stirring to force into forming aggregates	++	+++	++	±	+++	+++	++	++	+	++	+	Shear force from continuous spinning may affect cells. Require specific devices. May alter gene expression.	[43]
Rotating wall Vessel	A cylindrical vessel containing single-cell suspension creates a microgravity environment by constant horizontal rotation thus allowing cells to remain suspended and agitated enough to form spheroids	+	+	-	+	+++	+++	+++	++	++	++	+	May alter gene expression. Require specific	-
Magnetic Levitation	Cells are magnetized using magnetic nanoparticles and suspended in culture media using a magnetic force. The magnetic force also facilitates cell aggregation	++	+++	++	±	+++	+++	+++	++	+	++	+	Specific device arrangement and cell magnetization required	-
Microwell culture	Specially designed uniform microwells (i.e. low attachment microwell plate, microwells molded in agarose gel) (~500–1000 μm sized) with low attachment surface allow cells to remain suspended in culture media inside a compact space	++	+++	++	+++	++	+++	+++	±	+	++	+	Specialized molding and microarray patterning is required	[44]
Hydrogel embedding	ECM mimetic temperature controllable hydrogels (i.e. Matrigel, Geltrex, Collagen) are used to entrap cancer cells in an ECM like an environment that facilitates cancer cell organization into spheroids in a native-like environment	±	±	++	±	±	++	+++	-	±	+++	++	Can mimic in-vivo ECM and preserve TME Suitable for a wide range of cell types Difficult harvesting process may harm cells	[45]
Non-hydrogel scaffolds	Decellularized organ scaffolds or synthetic fibrous scaffolds provide porous surfaces for cells to get adsorbed and form 3D micro-tissue-like structures	±	+	±	-	-	++	±	+	±	+++	++	Mimic in-vivo ECM structure High cell-surface ratio Protect cells from the outside environment. Harvesting of spheroids can be difficult	[46]
ALI	Cells embedded in ECM hydrogel are placed on the upper surface of cell culture inserts exposed to air and get nutrition from culture media below through the porous membrane of the insert	±	++	+	-	+++	++	++	-	-	++	++	Suitable for long-term culture and invasion assay Can incorporate stromal and epithelial components Vulnerable to external environment	[47]
Microfluidic Device (Organ-on-a-chip)	Cells are dispersed into specially designed chip that contains microchambers and channels that can harbor cell suspensions, and hydrogels and dynamically deliver nutrients, drugs, or other components.	±	±	-	+	-	++	++	+	++	+++	+++	Dynamic environment Can simulate a wide range of physiological processes Can mimic organ structure and function Require specific device Require skilled handling	[48]
Bioprinting	Cells in bio-ink (composed of media, ECM material, hydrogel, microcarriers, etc.) are deposited layer-by-layer in a computer-aided designed pattern to generate viable 3D structures	±	+	-	++	++	+++	+++	+	+++	+++	+++	Better spatial control of different cells and components Can simulate a wide range of physiological processes Can mimic organ structure and function Require specific device Require skilled handling	[49]

ECM= Extra-cellular matrix, TME= Tumor microenvironment, ALI= Air-liquid interface.

Figure 1. Different cell culture techniques to form spheroids from cancer cells. (a) Hanging drop: gravitational pull facilitates spheroid formation (b) suspension culture: plate surface modification prevents attachment and facilitates cell aggregation (c) high viscosity suspension: media additives (e.g. methyl cellulose) increase viscosity and facilitate cell aggregation (d) pellet culture: centrifugation assists spheroid formation (e) magnetic levitation: magnetized cells aggregates to form spheroid assisted by a magnetic field (f) spinner flask: continuous agitation prevents cell attachment and facilitate spheroid formation (g) microwell plate: micropatterned wells allow uniformly sized spheroid formation (h) hydrogel embedding: hydrogel containing extracellular matrix components (laminin, collagen, etc.) aid in spheroid formation (i) fibrous scaffold: decellularized or synthetic fibrous scaffold support spheroid formation in-vitro (j) bioprinting: cells within bio ink layered in computer-aided patterns supports the formation of 3d structures (k) tumor-on-a-chip: specially designed chips containing microwells and channels supports spheroid growth with dynamically controlled environment. The illustration is created with BioRender.com.

Figure 2. Multiple methods of ICB efficacy assessment from spheroid-PBMC co-culture. (a) Cocultured spheroids are harvested and dissociated for flow cytometric analysis of T-Cells to compare activated T-cell population and tumor cell population levels (b) spheroids are stained with live-dead fluorescent staining and imaged to compare the proportions of dead cells between treatment and control (c) co-cultured spheroids are fixed and fluorescently labeled for imaging analysis where the spatial distribution of immune cells is compared (d) co-cultured spheroids are treated with ATP based bioluminescence assay for viability analysis (e) co-cultured well substrates are analyzed by ELISA method to compare levels of CD8+ activation enzymes e.g. Granzyme B. The illustration is created with BioRender.com.

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