Advanced 3D Tumor-Immune Co-Culture Assays
ProNAMs provides 3D tumor–immune co-culture assay services for immuno-oncology research. Patient-derived 3D tumor microtissues and 3D TME models are combined with defined immune effector populations to generate functional data on immune-mediated killing, cytokine response, and immune cell phenotype under controlled in vitro conditions.
Why 3D Co-Culture for Immuno-Oncology
A tumor is not a uniform mass of malignant cells but an ecosystem in which tumor, stromal and immune compartments interact. Preclinical models have historically captured this unevenly:
Traditional Preclinical Models
The Clinical Reality
Our co-culture platform reconstructs key elements of the human TME in vitro, providing a controlled system for mechanistic studies and for screening of immunotherapeutic candidates in a human-relevant context.
Our Platform Features
Two Assay Strategies
According to your research goals, there are two co-culture formats available for selection.
| Endogenous-Immune Format | Reconstitution Format | |
|---|---|---|
| Principle | Tumor tissue fragments are cultured in an air-exposed 3D format that retains immune and stromal cells already present in the sample | Purified 3D tumor microtissues are established first, then defined exogenous immune populations are introduced |
| Immune Source | Endogenous: native TILs, macrophages, and stromal fibroblasts carried over from the tissue | Exogenous: PBMCs, expanded TILs, isolated subsets, or engineered effector cells |
| Composition Control | Limited: reflects what was present in the sample | Defined: the subset identity, purity and E:T ratio are set by the study design |
| Consistency Across Arms | Lower: composition varies between donors and within a sample | Higher: the same effector preparation can be applied across all arms |
| Throughput | Low | Higher: compatible with plate-based screening formats |
| Usable Window | Limited: endogenous immune composition and viability shift over time in culture, which constrains long assays and repeat sampling | Extended: effector cells can be replenished |
| Material Required | Fresh resected tissue, processed within a defined window of collection | Established 3D model plus a separate immune cell source |
| Best Suited to | Questions about the composition and response of the TME as it arrived; exploratory profiling across donors | Mechanistic studies, dose–response work, compound screening, cell therapy potency comparison |
Both formats are subject to the same limitation: any ex vivo culture alters the tissue relative to its original state. The endogenous-immune format preserves more of the original cellular composition than dissociation-based approaches, but the composition still shifts over the culture period.
Immune Cell Co-Culture Modules
T Cell Co-Culture (CD8⁺, CD4⁺, CAR-T, TCR-T)
| Application | Description |
|---|---|
| Checkpoint Inhibitor Evaluation | PD-1/PD-L1 or other checkpoint-targeting antibodies introduced to assess effects on T cell activation and tumor structure loss |
| Adoptive Cell Therapy Assessment | Penetration and cytotoxicity of CAR-T or TCR-T products against 3D tumor structures with intact matrix barriers, including origins where matrix density is a known obstacle |
| Tumor-Reactive T Cell Expansion | 3D tumor microtissues used as a source of tumor antigen for ex vivo expansion of reactive T cells from peripheral blood |
Readouts: proliferation (CFSE dilution or Ki67), degranulation (CD107a), cytokine profiling (IFN-γ, TNF-α, IL-2), cytotoxic effector proteins (granzyme B, perforin), exhaustion marker phenotyping (PD-1, TIM-3, LAG-3, TIGIT).
Macrophage / TAM Co-Culture
Tumor-associated macrophages frequently adopt pro-tumor phenotypes, and macrophage polarization is sensitive to culture conditions. The 3D format provides oxygen and metabolite gradients closer to the tumor context than monolayer culture, which supports investigation of polarization under more representative conditions.
| Application | Description |
|---|---|
| Polarization Mechanisms | How tumor-derived soluble factors and 3D context drive macrophage phenotype |
| Phagocytosis-Directed Therapies | Evaluation of agents such as CD47-axis antibodies or CSF-1R inhibitors |
| Resistance Mechanisms | How macrophage-derived immunosuppressive factors (IL-10, TGF-β) and physical positioning affect T cell access to tumor structures |
Readouts: surface marker phenotyping, phagocytosis quantification by imaging or flow, secreted factor profiling, and effects on co-cultured T cell function.
Natural Killer (NK) Cell Co-Culture
NK-mediated killing does not require MHC-restricted antigen presentation, which makes allogeneic healthy-donor NK cells a practical effector source for co-culture with patient-derived 3D tumor models. KIR–HLA compatibility between donor and target is a recognized modifier of NK activity, and donor selection or KIR genotyping can be incorporated into the design where it is material to the question.
| Application | Description |
|---|---|
| ADCC Evaluation | Tumor-targeting monoclonal antibodies introduced to quantify antibody-dependent cellular cytotoxicity against 3D structures |
| Allogeneic Cell Therapy Assessment | Potency evaluation of CAR-NK and related products against solid tumor 3D models |
| Combination Strategies | Whether chemotherapy or radiation upregulates NKG2D ligands on tumor structures and sensitizes them to NK killing; whether cytokine support such as IL-15 improves penetration into 3D structures |
Readouts: killing quantification, degranulation, cytokine profiling, NK activating and inhibitory receptor phenotyping, target ligand expression.
Dendritic Cell (DC) Co-Culture
| Application | Description |
|---|---|
| Tumor Vaccine Development | Whether 3D tumor microtissue lysates or defined neoantigens drive DC maturation and subsequent antigen presentation |
| Antigen Presentation Modeling | How tumor-derived TGF-β and other factors suppress DC maturation in a 3D context |
| DC-Directed Checkpoint Regulation | DCs express regulatory receptors including PD-L1, TIM-3 and CD80/86. Blocking antibodies directed at these can be introduced to assess effects on DC maturation and cross-presentation, and on downstream T cell priming in three-way co-culture |
Readouts: maturation marker phenotyping, cytokine profiling, antigen uptake and processing, downstream T cell activation in DC-T-tumor configurations.
Assay Parameters & Readouts
Parameters are set per study and the ranges below indicate typical starting points confirmed during design.
| Parameter | Typical Configuration |
|---|---|
| Effector-To-Target Ratio | Varies substantially by effector type and endpoint - from low ratios for extended imaging-based assays to high ratios for short-duration cytotoxicity endpoints. Established per effector type and tumor origin during design |
| Co-Culture Duration | Commonly 24–96 hours for cytotoxicity and cytokine endpoints; extended schedules used for expansion and repeat-stimulation designs |
| Effector Preparation | Fresh or thawed PBMCs, with or without prior activation; expanded TILs; isolated subsets; client-supplied engineered effector cells |
| Replicates | Commonly three or more independent wells per condition; final number specified in the protocol |
| Controls | Tumor structures alone, effector cells alone, isotype-matched antibody control, and a positive cytotoxicity control appropriate to the effector type |
To comprehensive evaluate therapeutic efficacy, our platform integrates multi-dimensional profiling across cellular, spatial, and molecular levels.
| Readout Category | Methods |
|---|---|
| Cytotoxicity | Live-cell imaging with structure-level quantification, viability reagents adapted to 3D format, apoptosis-specific ELISA |
| Cytokine and soluble factors | ELISA, multiplex bead-based assays |
| Immune cell phenotype | Flow cytometry - activation, exhaustion, differentiation, and lineage panels |
| Spatial analysis | Immunofluorescence imaging of infiltration and localization relative to tumor structures |
| Molecular | RT-qPCR; transcriptomic analysis where the design calls for it |
Service Workflow
| Stage | Activity | Client Input | Indicative Duration |
|---|---|---|---|
| 1. Scoping | Consultation to define the biological question, assay format, effector type, tumor origin, and endpoints | Research objective, test article class, target and mechanism | 3–5 business days |
| 2. Design & Quotation | Written protocol covering E:T strategy, co-culture duration, controls, readouts, replicate number, and statistical approach | Protocol review and sign-off | 5–10 business days |
| 3. Material Preparation | Establishment or recovery of 3D tumor models to assay-ready quantity; sourcing or receipt of effector cells | Test article; client-supplied cells or models where applicable | Highly variable by tumor origin; commonly 4–10 weeks |
| 4. Assay Feasibility | Small-scale run to establish working E:T ratio, confirm assay window, and verify readout performance for the specific model–effector pairing | - | 2–3 weeks |
| 5. Study Execution | Full-scale co-culture, dosing, sampling, and endpoint acquisition | - | Assay phase commonly under 1 week for cytotoxicity and cytokine endpoints; longer for expansion or repeat-stimulation designs |
| 6. Analysis & Reporting | Data analysis, statistical evaluation, draft report, client review round, final report | Comments on draft report | 10–15 business days |
Browse Deliverables & Timelines
NOTE: All timelines, quantities and deliverables described on this page are indicative.
Frequently Asked Questions
It depends on the endpoint. Allogeneic T cell configurations carry a background problem: donor T cells recognize mismatched MHC on the tumor structures as foreign, producing killing that is unrelated to the mechanism under test and that reduces assay specificity. Where antigen-specific T cell recognition is the endpoint, an autologous or HLA-matched configuration is needed. NK-based assays are less constrained in this respect, since NK killing is not MHC-restricted, though KIR–HLA relationships still modify activity and are accounted for in donor selection. Autologous configurations require matched material and extend the timeline substantially, so the design decision is made on whether the endpoint requires it.
The usable window is limited and varies by tissue. Immune composition and viability shift over the culture period, and published assessments of this class of model note that variation in cell composition and short-term viability constrain its use for high-throughput screening. Assay windows are fixed during design based on the tumor origin, and the format is generally applied to shorter, exploratory studies rather than to extended screening campaigns.
The reconstitution format is compatible with plate-based screening. Scaling requires that the assay window, E:T ratio and readout be established first for the specific model–effector pairing, which is the purpose of the Stage 4 feasibility run. The endogenous-immune format is not suited to screening at scale for the reasons above.
Not in every case. Detection of tumor-reactive responses in autologous systems depends on factors including the mutational burden of the tumor and its antigen presentation status; published studies report reactivity in a subset of donor–tumor pairs rather than uniformly. Study designs address this by including appropriate controls and, where the question allows, by testing across multiple donors. Where a specific reactivity threshold is required for the program, this is discussed at Stage 1 so that the design and the interpretation of a negative result are agreed in advance.
Assays developed for monolayer cultures do not transfer directly. Approaches used include image-based quantification of tumor structure integrity over time and apoptosis-specific ELISA detecting epithelial cell death markers. Method selection depends on the tumor origin, the effector type and the required sensitivity, and is fixed in the protocol before the study begins.
Both are possible. We can source allogeneic healthy-donor PBMCs or NK cells, isolate defined subsets, and expand TILs where matched tissue is available. Clients frequently supply their own engineered effector products such as CAR-T or CAR-NK preparations, in which case receipt requirements and biosafety documentation are agreed at Stage 2.
Discuss Your Immuno-Oncology Study
Contact our technical team for a project assessment covering assay format, effector source, endpoint selection, and indicative timeline.
Schedule a Consultation