In vitro immuno-oncology (IO) therapeutics testing is used to evaluate the efficacy, mechanism of action, and resistance to cancer immunotherapies before they advance into more costly preclinical and clinical development. By co-culturing 3D tumor spheroids with Peripheral Blood Mononuclear Cells (PBMCs) or specific immune cell subpopulations such as T cells, NK cells, or macrophages, it is possible to investigate immune-cell activation, tumor killing, cytokine release, immune-cell infiltration, and IO treatment efficacy within a physiologically relevant solid tumor-like microenvironment.
Targeted IO therapies have reshaped cancer treatment, marked by the approval of the first immune checkpoint inhibitor in 2011 and the PD-1 inhibitor Nivolumab in 2014. Although immune checkpoint inhibitors have significantly improved clinical outcomes across multiple cancer types, only 20–40% of patients achieve durable responses (He & Xu, 2020, Morad et al., 2021). Recent clinical setbacks of IO therapies targeting TIGIT, TIM-3, and TGF-β further highlight the challenge of translating promising preclinical findings into clinical success (Roche, 2024; GSK, 2024; Merck KGaA, 2021). These limitations underscore the need for predictive biomarkers and human-relevant in vitro models that better recapitulate the biological complexity of the tumor microenvironment, improve prediction of treatment efficacy, and support patient stratification for immunotherapy.
InSphero’s 3D InSight™ Immuno-Oncology Platform enables translational immuno-oncology research using human 3D tumor spheroids co-cultured with PBMCs or specific immune cells, recreating key features of the tumor microenvironment. Our platform supports comprehensive evaluation of IO therapy efficacy, mechanism of action, immune-cell infiltration, cytokine release or resistance mechanisms in a more physiologically relevant setting than conventional 2D in vitro models and assays. Thus, enabling researchers to generate more predictive data and make informed decisions from early discovery through preclinical development.
The Challenge with Conventional 2D In Vitro Models
While conventional 2D cell cultures remain valuable for early-stage screening and target validation, they do not adequately capture the complexity of the tumor microenvironment found in solid tumors.
Key limitations include:
These limitations explain why IO therapies that demonstrate promising activity in traditional 2D in vitro models can still show limited efficacy in patients.
The Advantage of 3D Tumor Spheroids
3D tumor spheroids replicate key aspects of the in vivo tumor microenvironment, enabling a more translationally relevant evaluation of immuno-oncology therapies.
Benefits of 3D tumor models include:
By better reflecting the biological complexity of solid tumors, 3D tumor spheroids allow the generation of more physiologically relevant insights into mechanisms of action, efficacy, and resistance before advancing candidates into costly in vivo studies and clinical development.
Building on the physiological relevance of 3D tumor spheroids, our 3D InSight™ Immuno-Oncology Platform combines tumor models, immune-cell co-cultures, and multiparametric readouts to help you evaluate immunotherapy efficacy and mechanism of action with greater translational relevance – supporting more confident decisions from early discovery through preclinical development for next-generation immune-oncology therapies.
Generate physiologically relevant data on the mechanism of action, efficacy, and resistance mechanisms to support confident IO therapy development decisions.
Evaluate IO therapies in 3D tumor models that capture key features of solid tumors, including tumor- immune cell or tumor-stroma interactions and spatial organization.
Co-culture tumor spheroids with PBMCs or specific immune cell subpopulations such as T cells, Natural Killer (NK) cells or macrophages to investigate immune activation, infiltration, and anti-tumor activity.
Connect immune activation to therapeutic outcomes through multiparametric endpoints, including tumor killing, immune infiltration, cytokine release, apoptosis, and phagocytosis.
Evaluate immune checkpoint inhibitors, T-cell engagers, bispecific antibodies, CAR-T therapies, macrophage-targeting agents, combination therapies, and novel immuno-oncology modalities within a single translational platform.
Our 3D InSight™ Immuno-Oncology Platform supports the evaluation of multiple immuno-oncology modalities within physiologically relevant tumor microenvironments.
Multispecific antibodies including bispecifics and emerging trispecific antibodies, such as T-cell engagers (TCEs), rely on efficient immune-cell recruitment and close interaction between immune and tumor cells. Their activity can be strongly influenced by the spatial organization and penetration barriers found in solid tumors (Labrijn et al, 2019).
Evaluate: The ability of multispecific antibodies to recruit immune cells, trigger immune activation, and induce tumor killing within 3D tumor models that mimic the spatial complexity of solid tumors.
Although immune checkpoint inhibitors (ICIs) have transformed cancer treatment, many tumors remain resistant due to immune exclusion and suppressive tumor biology (Wang et al., 2023; Morad et al., 2021).
Evaluate: How checkpoint inhibitors modulate immune responses within a physiologically relevant tumor microenvironment by assessing immune-cell activation, infiltration, tumor killing, and resistance mechanisms that may influence therapeutic efficacy in solid tumors.
The efficacy of CAR-T and other cell-based therapies in solid tumors is often limited by immune exclusion, stromal barriers, and immunosuppressive signaling, which restrict immune-cell infiltration and anti-tumor activity (Newick et al., 2017).
Evaluate: Immune-cell infiltration, activation, and anti-tumor activity in the presence of physiologically relevant barriers such as immune exclusion and suppressive tumor microenvironment signalling.
Combination immunotherapies are increasingly used to overcome resistance mechanisms associated with immune suppression, stromal-mediated exclusion, hypoxia, and limited immune-cell activity within solid tumors (Morad et al., 2021)
Evaluate: Combination strategies that can enhance immune activation, overcome resistance mechanisms, improve immune-cell infiltration, and drive stronger anti-tumor responses compared to monotherapies.
Answer critical questions across your immuno-oncology program – from immune activation and tumor killing to resistance mechanisms with comprehensive assays built on physiologically relevant 3D tumor models.
Does your IO therapy induce meaningful anti-tumor activity?
Monitor tumor growth and immune-mediated tumor killing in 3D tumor spheroids co-cultured with PBMCs to quantify therapeutic efficacy over time and multiplex with downstream mechanistic endpoints.
Key readouts:
How effectively does your IO therapy eliminate tumor cells?
Quantify viable and dead cells within 3D tumor spheroids using fluorescence-based live/dead staining to assess treatment-induced cytotoxicity.
Key readouts:
How does your therapy modulate immune responses?
Characterize immune activation and inflammatory signalling by analysing and quantifying cytokines and cytotoxic effector molecules released following treatment using a 13-plex human cytokine panel.
Key readouts:
How are tumor cells being eliminated?
Investigate intracellular pathways involved in immune-mediated tumor killing and apoptosis induction.
Key readouts:
Can immune cells penetrate and engage the tumor?
Assess immune-cell infiltration into 3D tumor spheroids and identify the immune populations driving anti-tumor responses by applying high-content imaging at single-cell resolution.
Key readouts:
Can macrophages recognize and eliminate tumor cells?
Evaluate macrophage-mediated tumor clearance and the impact of innate immune checkpoint modulation.
Key readouts:
From biological questions to development decisions, our scientists collaborate with your team to design and execute tailored immuno-oncology studies that generate actionable, translational insights for your program.
Tell us about your immuno-oncology therapy, biological question, and development objectives. Our scientists will help define the most relevant model and study strategy.
Together, we build a tailored study using the appropriate 3D tumor model, immune-cell co-culture system, and assay readouts.
Our experts execute the study and generate functional and mechanistic data on therapeutic activity, immune responses, and tumor microenvironment-associated resistance mechanisms.
Receive a comprehensive study report and discuss the findings with our scientists to support candidate prioritization and future development decisions.
2D tumor models are useful for early screening and target validation, but they do not fully capture tumor architecture, spatial organization, immune exclusion, penetration barriers, or tumor microenvironment-associated resistance. 3D tumor spheroids provide a more solid tumor-like architecture for evaluating immune-tumor interactions and evaluating mechanism of action and efficacy of IO therapeutics.
Using 3D tumor spheroid models one can evaluate various IO therapies including checkpoint inhibitors, T-cell engagers, bispecific antibodies, CAR-T and other cell therapies, macrophage-based therapies, combination therapies, to novel immuno-oncology modalities.
Relevant readouts may include tumor growth kinetics, immune-mediated tumor killing, live/dead staining, immune cell infiltration, cytokine release, apoptosis markers such as cleaved caspase-3/7, and macrophage phagocytosis.
3D InSightTM Immuno-Oncology Platform can accelerate human-relevant, predictive early discovery and preclinical development of IO therapies. 3D tumor spheroids and assays support generation of data into mechanism-of-action, efficacy, resistance investigation, supporting confident go/no-go decisions before more complex in vivo or clinical studies.
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