Protocol to develop a 3D mouse autologous lung tumor-immune spheroid co-culture model to advance radiotherapy-based treatments

Summary

3D culture models better replicate tumor structure and interactions than 2D methods, enhancing cancer therapy research. Here, we present a protocol to develop a 3D mouse autologous lung tumor-immune spheroid co-culture model to advance radiotherapy-based treatments. We describe steps for optimizing tumor spheroids, isolating CD4+ T cells and monocytes, and differentiating T regulatory (Treg) cells and M2-like macrophages. We then detail procedures for seeding tumor-immune spheroids, performing fluorescence-activated cell sorting (FACS) analysis, and characterizing spheroids, including their response to radiotherapy.

Graphical abstract

Highlights

• •Optimization of a 3D culture technique based on TSp formation in agarose molds
• •Enrichment of TSp with T regulatory cells and M2-like macrophages
• •Characterization of TImS using cell trackers and flow cytometry
• •Assessment of radiotherapy efficacy in TImS in a unique autologous lung cancer model

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

3D culture models better replicate tumor structure and interactions than 2D methods, enhancing cancer therapy research. Here, we present a protocol to develop a 3D mouse autologous lung tumor-immune spheroid co-culture model to advance radiotherapy-based treatments. We describe steps for optimizing tumor spheroids, isolating CD4+ T cells and monocytes, and differentiating T regulatory (Treg) cells and M2-like macrophages. We then detail procedures for seeding tumor-immune spheroids, performing fluorescence-activated cell sorting (FACS) analysis, and characterizing spheroids, including their response to radiotherapy.

Before you begin

In this protocol, we first optimize the development of Lacun3¹ and CMT167² TSp in standard culture conditions. This is an important step before conducting the TImS. In our case, TSp containing different cell numbers (1,000, 2,500 and 5,000 cells) are tested. To decide the proper cell-size we assess cell proliferation rate, cell viability assay (Flow cytometry) and measurement of mitochondrial activity of each TSp. Additionally, we take photographs at days 1, 3, 6 and 8 of experiment to determine the diameter, circularity and compactness of TSp.

Institutional permissions

All animal experiments were performed under strict recommendations from the European Union Directive 2010/63/EU, following a protocol previously approved by the i3S Ethics Committee and Direção Geral da Alimentação e Veterinaria (DGAV) (reference: BSm_2017_10) and by the Ethical Committee for the experimentation with animals of the University of Navarra (ref. 082-23). At the defined endpoints, mice were euthanized by cervical dislocation by trained personnel. Animals were kept in a pathogen-free environment at CIMA or i3S animal facility.

Optimization for TSp

Timing: 4 weeks

Here, we outline the essential steps for optimizing TSp. The number of tumor cells should be adjusted based on the specific experimental conditions and the cell line used. Ideally, cell viability at the endpoint of the experiment should be approximately 80%. We recommend starting the optimization process with a range of 1,000 to 2,500 cells per mold.

• 1.
  Use agarose molds from MicroTissues 3D Petri Dish. The process to cast agarose molds is shown in Figures 1A–1C. Each MicroTissues 3D Petri Dish consists of 81 wells, 9 × 9 well array.

  • a.
    Autoclave separately 200 mL of saline solution (0.9% NaCl in water) and agarose powder (4 g).
  • b.
    Mix the saline solution and the agarose in sterile conditions.
  • c.
    Heat the mix in the microwave until it is completely melted.
  • d.
    Add 600 μL of 2% agarose to each MicroTissues 3D Petri Dish.
  • e.
    Wait for 5 min until complete solidification of the agarose.
  • f.
    Introduce each agarose mold in individual wells of 12-well culture plates by inverting the MicroTissues 3D Petri Dish and gently allowing it to drop into each well.
  • g.
    Fill the well up to 2 mL of complete medium (RPMI or DMEM) to allow the hydration of the mold.

Note: The hydration of the mold needs to be done at least 2 h before cell seeding inside the agarose molds.

• 2.
  Collect tumor cells.

  • a.
    Aspirate supernatant from flasks.
  • b.
    Add 5 mL of 0.05% Trypsin-EDTA and keep the cells in the humified incubator for 5 min (37°C and 5% CO₂).
  • c.
    Neutralize the trypsin with 5 mL of complete medium (complete RPMI for Lacun3 and complete DMEM for CMT167) and collect the cells into 15 mL centrifuge tubes.
  • d.
    Pellet the cells by centrifuging at 310 rcf for 5 min at room temperature.
  • e.
    Discard supernatant and resuspend the cells in 3 mL of corresponding complete medium. Ensure a homogenous unicellular suspension.
  • f.
    Determine the number of cells preparing a 1:10 mix of cell suspension in Trypan Blue staining. Use a cell counter.

    CRITICAL: In this case, as an example, we calculate the number of cells for two molds per condition. To calculate the total number of cells that we need for each mix, we consider 81 wells (9 × 9 well array) per mold and three molds per condition (accounting for 1 mold of excess). We prepare 6 different mixes:

  • g.
    Assay conditions and calculations:

    • i.
      Lacun3 # 1,000 cells – $1,000cells\ast 81wells\ast 3molds=243,000cells$
    • ii.
      Lacun3 # 2,500 cells – $2,500cells\ast 81wells\ast 3molds=607,500cells$
    • iii.
      Lacun3 # 5,000 cells – $5,000cells\ast 81wells\ast 3molds=1,215,000cells$
    • iv.
      CMT167 # 1,000 cells – $1,000cells\ast 81wells\ast 3molds=243,000cells$
    • v.
      CMT167 # 2,500 cells – $2,500cells\ast 81wells\ast 3molds=607,500cells$
    • vi.
      CMT167 # 5,000 cells – $5,000cells\ast 81wells\ast 3molds=1,215,000cells$
• 3.Prepare 3 different mixes per cell line.

Note: 190 μL of each cell suspension per mold are added. In this case we need $190\mu L\ast 3molds=570\mu L$ per mix. If the volume of cells needed is less than the required for each mix, we recommend adding the remaining volume of media up to 570 μL and homogenize the cell suspension. If the volume of cells is more than 570 μL, cells have to be pelleted (310 rcf for 5 min), the supernatant carefully removed and resuspended up to the corresponding volume of medium (570 μL in this example).

• 4.
  Collect all the medium from each well into a 50 mL centrifuge tube. This medium can be used to fill up the wells in the step 7.

  • a.
    Aspirate medium from the outside of the mold with a Pasteur pipette.
  • b.
    Carefully collect the medium from the inside of the mold. Use a 200 μL pipette from the corner of the agarose mold.
• 5.Add 190 μL of cell suspension mix per mold.
• 6.Keep the plate inside the incubator and wait for 30 min to let the spheroids settle at the bottom of the molds.
• 7.Fill the well of the plate up to 2 mL with the corresponding complete medium collected in the step 4.
• 8.Place the plate in the incubator.

Note: After step 8, we continue with the functional characterization of TSp. Photographs of the TSp are taken at days 1, 3, 6 and 8 (Step 9). Take as many images as desired, with a minimum of 4 spheres per condition. Viability (Step 10) and mitochondrial activity (Step 11) are measured at days 3 and 8 of experiment. Importantly, we use all the spheroids from each mold to measure mitochondrial activity and viability, thus, different plates are required for each experiment. However, after taking photos, plates are placed in the incubator. An overview of the process is shown in Figure 1D.

Note: Seeding conditions are primarily determined based on cell viability at day 8, which should ideally be approximately 80%. Additionally, the proliferation rate and mitochondrial activity of both cell lines should be comparable by the end of the experiment.

• 9.Take images of TSp at days 1, 3, 6 and 8 of experiment (Figure 2). A Primovert microscope and Labscope App (ZEISS) is used to take photographs of four randomly selected TSp per condition (Figure 2A).
• 10.Use AnaSP software³ to calculate the diameter, circularity, and compactness of each TSp, following the guidelines recommended by the MISpheroID consortium⁴ (Figures 2B and 2C).
• 11.
  Calculate the cell viability of the TSp at days 3 and 8 of experiment (Figure 3A).

  • a.
    Collect the TSp from the mold and place it into a 15 mL centrifuge tube:

    • i.
      Remove 1 mL from the outside of the well.
    • ii.
      Collect all the TSP along with the remaining medium by gently pipetting “up and down” several times within the agarose mold to ensure all TSP are gathered.
    • iii.
      Use PBS if needed to collect all the TSp of the mold. Repeat this process as necessary.
  • b.
    Centrifuge at 480 rcf for 5 min at room temperature.
  • c.
    Remove supernatant and wash the TSp with 2 mL of PBS.
  • d.
    Centrifuge at 480 rcf for 5 min at room temperature.
  • e.
    Discard all the supernatant.
  • f.
    Use 300 μL of TrypLE^TM Select 2× (prepared in PBS) to dissociate the TSp. Mix carefully.
  • g.
    Keep the TSp at 37°C for 20 min.

    Note: This incubation might be different depending on the cells included in the spheroids. It is recommended to test different concentrations of TrypLE and / or increase the incubation time.

    Note: The following steps are performed at 4°C. Keep the PBS and FACS buffer on ice and refrigerate the centrifuge at 4°C.

  • h.
    Add 2 mL of cold PBS and mix roughly.
  • i.
    Centrifuge at 480 rcf for 5 min at 4°C.
  • j.
    Carefully discard the PBS.
  • k.
    Resuspend in 200 μL of FACS buffer and transfer into a v-bottom 96-well plate.
  • l.
    Centrifuge the plate at 480 rcf for 5 min.
  • m.
    Carefully discard the supernatant by inverting the plate.
  • n.
    Perform the viability staining:

    • i.
      Add 100 μL of mix for viability staining.
    • ii.
      Incubate 30 min at 4°C in darkness.
    • iii.
      Add 100 μL of FACS Buffer to improve cell recovery after centrifugation.
    • iv.
      Centrifuge at 480 rcf for 5 min at 4°C.
    • v.
      Carefully discard the supernatant.
  • o.
    Resuspend the cells in 200 μL of FACS Buffer.
  • p.
    Add 10 μL of Precision Count Beads (1000 beads/μL) and mix thoroughly to calculate the proliferation capability of tumor cells (Figure 3B). To calculate the proliferation next formula was applied:

    $$Absolutecellcount\left(\frac{cells}{\mu L}\right)=\frac{Cellcount}{Precissioncountbeadscount}xprecissioncountbeadsconcentration\left(\frac{beads}{\mu L}\right)$$
  • q.
    Acquire samples in the CytoFlex LX or similar Flow Cytometer and analyze the data with CytExpert software.
• 12.
  Measure the mitochondrial activity of the spheroids at days 3 and 8 of experiment (Figure 3C).

  CRITICAL: Be careful when replacing the medium from each well of the plate. Aspirate the medium from the outside of the mold with a Pasteur pipette.

  • a.
    Carefully remove medium from wells by aspirating with a Pasteur pipette.
  • b.
    Add 2 mL of culture media containing 20% of resazurin (v/v).
  • c.
    Incubate the plate for 2 h at 37°C, in darkness.
  • d.
    Transfer 200 μL of medium from each well (in triplicates) to a black-walled, clear-bottom 96-well plate.
  • e.
    Measure the fluorescence at the excitation and emission wavelengths of 530 nm and 590 nm, respectively, using a GloMax Discover Microplate reader (or similar).

Figure 1.

Casting process of agarose molds and functional characterization of murine lung TSp

(A) Heat agarose in the microwave and prepare MicroTissues 3D Petri Dish. Add 600 μL of melted agarose and wait until complete solidification.

(B) Introduce each agarose molds in individual wells of 12-well culture plate.

(C) Fill the well up to 2 mL of complete medium. Place the culture plate in a humidified incubator until seeding TSp or TImS. Hydration of the molds needs to be done at least 2h before seeding.

(D) Diagram summarizing the functional characterization assays of cells within murine lung TSp. Plate #1 is used to measure cell viability while plate #2 is used to measure mitochondrial activity.

Figure 2.

Morphological and physical characterization of TSp

(A) Representative images of TSp formation using different cell number seeding (1,000, 2,500, and 5,000 cells). Images were taken on days 1, 3, 6, and 8, for Lacun3 and CMT167 lung cancer cells.

(B) Pipeline followed in AnaSP software to analyze diameter, compactness and circularity of different spheroids. We first create a mask of the spheroid. Then the software automatically calculates different parameters of each TSp.

(C) Diameter, compactness, and circularity parameters were used to characterize the physical properties of TSp. AnaSP software was employed for the analysis. Data are expressed as mean ± SD and were analyzed with a two-way ANOVA followed by a Bonferroni test. ∗: p <0.05; ∗∗: p <0.01; ∗∗∗: p <0.001.

Figure 3.

Functional characterization of TSp

Graphs showing cell viability (A) proliferation rate (B) and mitochondrial activity (expressed in MFI) (C) of Lacun3 or CMT167 TSp. Data are expressed as mean ± SD and were analyzed with student’s t-test. ∗: p <0.05; ∗∗: p <0.01.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
AF700 rat anti-mouse CD11b (clone M1/70)	BioLegend	Ref: 101222 / RRID: AB_493705
Alexa Fluor 488 anti-mouse Ly-6C (clone HK1.4)	BioLegend	Ref: 128006 / RRID: AB_1186135
APC/Fire 750 anti-mouse CD45 (clone 30-F11)	BioLegend	Ref: 103154 / RRID: AB_2572116
Brilliant Violet 421 anti-mouse F4/80 (clone BM8)	BioLegend	Ref: 123132 / RRID: AB_11203717
Brilliant Violet 510 anti-mouse/human CD11b (clone M1/70)	BioLegend	Ref: 101245 / RRID: AB_2561390
Brilliant Violet 650 anti-mouse CD206 (clone C068C2)	BioLegend	Ref: 141723 / RRID: AB_2562445
eFluor450 rat anti-mouse CD4 (clone RM4-5)	eBioscience	Ref: 48-0042-82 / RRID: AB_1272194
Pe/Cy7 anti-mouse Foxp3 (clone 3G3)	Abcam	Ref: ab210232
PerCP anti-mouse CD25 (clone PC61)	BioLegend	Ref: 102027 / RRID: AB_893290
Purified NA/LE hamster anti-mouse CD3e (clone 145-2C11)	BD Pharmingen	Ref: 553057 / RRID: AB_394590
ViaKrome 808 fixable viability dye	Beckman Coulter	Ref: C36628
Biological samples
Bone marrow	BALB/c and C57BL/6 mice	N/A
Spleen	BALB/c and C57BL/6 mice	N/A
Chemicals, peptides, and recombinant proteins
0.05% trypsin-EDTA	Gibco	Ref: 25300054
3.7%–4.0% p/v formaldehyde	PanReac AppliChem	Ref: 252931.1211
ACK lysing buffer	Gibco	Ref: A10492-01
Bovine serum albumin	Sigma	Ref: A3059
CellTrace CFSE cell proliferation	Thermo Fisher Scientific	Ref: C34554
CellTrace Far Red cell proliferation	Thermo Fisher Scientific	Ref: C34572
CellTrace Violet cell proliferation	Thermo Fisher Scientific	Ref: C34557
DMEM medium	Gibco	Ref: 41966-029
DPBS without calcium and magnesium	Corning	Ref: 21-031-CV
FetalClone III	Cytiva	Ref: 16281102
Fixation/permeabilization concentrate (4×)	Invitrogen	Ref: 00-5123-43
Fixation/permeabilization diluent	Invitrogen	Ref: 00-5223-56
HEPES buffer solution (1 M)	Gibco	Ref: 15630-056
HyClone Amphotericin B solution (Fungizone)	Cytiva	Ref: SV30078.01
Mouse IL-2	PeproTech	Ref: 212-12
M-CSF	MIltenyi	Ref: 130-101-706
MEM NEEA (100×)	Gibco	Ref: 11140-050
Penicillin-streptomycin (10,000 U/mL)	Gibco	Ref: 15140122
Permeabilization buffer	Invitrogen	Ref: 00-8333-56
Precision count beads	BioLegend	Ref: 424902
Recombinant human TGF-β1	InvivoGen	Ref: Rcyc-htgfb1
Recombinant murine IL-4	PeproTech	Ref: 214-14
Resazurin sodium salt	Merck	Ref: R7017
RPMI medium 1640	Gibco	Ref: 11875093
SeaKem LE agarose	Lonza	Ref: 50004
Sodium pyruvate (100 mM)	Gibco	Ref: 11360-070
Trypan blue	Sigma-Aldrich	Ref: T8154
TrypLE Select enzyme (10×), no phenol red	Gibco	Ref: A1217701
UltraComp eBeads	Invitrogen	Ref: 01-2222-41
Critical commercial assays
EasySep mouse monocyte isolation kit	STEMCELL	Ref: 19861
EasySep mouse naive CD4+ T cell isolation kit	STEMCELL	Ref: 19765
Foxp3/transcription factor staining buffer set	Invitrogen	Ref: 00-5523-00
Experimental models: Cell lines
CMT167 cell line	Paola Allavena’s Laboratory (Humanitas, Milan, Italy)	Anfray C et al.,
Lacun3 cell line	Luis Montuenga’s Laboratory (CIMA, Pamplona, Spain)	Freire J et al.,.
Experimental models: Organisms/strains
8-week-old female BALB/c mice	Envigo	N/A
8-week-old female C57BL/6 mice	Envigo	N/A
Software and algorithms
AnaSP	AnaSP, MATLAB software (version 2024-03-20)	https://sourceforge.net/projects/anasp/
BioRender	Biorender.com	https://www.biorender.com
CytExpert software	N/A	N/A
FlowJo v.10	version 10	N/A
GraphPad Prism 8.0.1	version 8	N/A
ZEN Blue	version 3.0	N/A
Other
BD LSRFortessa cell analyzer	BD Bioscience	N/A
CytoFlex LX	Beckman Coulter	N/A
GloMax Discover microplate reader	Promega	N/A
Synergy Mx multimode microplate reader SMA	BioTek	N/A
ZOE fluorescent cell imager	Bio-Rad	N/A
Primovert	Zeiss	N/A
Confocal laser scanning microscope LSM800	Zeiss	N/A
5 mL syringe	BD Emerald	Ref: 307731
V-bottom 96-well plate, non-treated, non-sterile	Thermo Scientific	Ref: 442587
96-well black polystyrene microplate	Corning	Ref: CLS 3631
96-well clear round bottom, non-treated microplate	Corning	Ref: 3879
Cell scraper	Sarstedt	Ref: 83.3951
Cell strainer (70 μm)	Falcon	Ref: 352350
EasySep magnet	STEMCELL	Ref: 18000
Glass bottom dish	WillCo-dish	GWST-5040
MicroTissues 3D Petri dish	Merck	Ref: Z764019
1.5 mL microcentrifuge tubes	Sarstedt	Ref: 72.690.001
0.2 mL PCR tubes	VWR	Ref: 732-0548
Sterile surgical blades	Braun	Ref: 16600584
Tissue culture plate, non-treated, 12 wells, sterilized	Avantor	Ref: 734-2778
Transfer pipette 3.5 mL	Sarstedt	Ref 86.1171.001
15 mL centrifuge tubes	Corning	Ref: 430052
50 mL centrifuge tubes	Corning	Ref: 430290

Materials and equipment

2% Agarose

Reagent	Final concentration	Amount
Water	N/A	200 mL
Sodium chloride (NaCl)	0.9%	1.8 g
Agarose	2%	4 g
Total	N/A	200 mL

Note: Saline solution (0.9% NaCl in water) and powder agarose (4 g) are autoclaved in different bottles and mixed inside the hood. Store at 25°C until use.

Complete RPMI medium
Reagent	Final concentration	Amount
RPMI medium 1640	N/A	445 mL
FetalClone III	10%	50 mL
Penicillin / Streptomycin	1%	5 mL
Total	N/A	500 mL

Note: Complete RPMI medium can be prepared in advance and can be stored at 4°C up to 1 month.

Complete DMEM medium

Reagent	Final concentration	Amount
DMEM medium	N/A	445 mL
FetalClone III	10%	50 mL
Penicillin / Streptomycin	1%	5 mL
Total	N/A	500 mL

Note: Complete DMEM medium can be prepared in advance and can be stored at 4°C up to 1 month.

CD4+ T cell culture medium

Reagent	Final concentration	Amount
Complete RPMI medium	N/A	483 mL
MEM NEEA	1×	5 mL
Fungizone	1 μg/mL	2 mL
HEPES	10 mM	5 mL
Sodium Pyruvate	1 mM	5 mL
Total	N/A	500 mL

Note: Complete medium for CD4 T cell culture can be prepared in advance and can be stored at 4°C up to 1 month.

FACS buffer

Reagent	Final concentration	Amount
PBS	N/A	485 mL
FetalClone III	2%	10 mL
Sodium azide	0.1%	5 mL
Total	N/A	500 mL

Note: FACS Buffer can be prepared in advance and can be stored at 4°C up to 1 month.

(2×) Treg differentiation medium (100 μL per well)

Reagent	Final concentration	Amount
Medium for CD4. T cell culture	N/A	97.6 μL
IL2	200 U/mL	2.22 μL
hTGFβ	10 ng/mL	0.2 μL
Total	N/A	100 μL

Note: Treg differentiation medium can be prepared in advance and can be stored at 4°C up to 1 month.

Macrophage differentiation medium (2 mL per well)

Reagent	Final concentration	Amount
Complete RPMI medium	N/A	12 mL
M-CSF	25 ng/mL	1.2 μL
Total	N/A	12 mL

Note: Macrophage differentiation medium can be prepared in advance and can be stored at 4°C up to 1 month.

M2-like macrophage differentiation medium (2 mL per well)

Reagent	Final concentration	Amount
Complete RPMI medium	N/A	12 mL
IL4	100 U/mL	1.2 μL
Total	N/A	12 mL

Note: M2-like macrophage differentiation medium can be prepared in advance and can be stored at 4°C up to 1 month.

Mix for extracellular staining (100 μL per sample)

Antibody	Clone	Fluorochrome	Dilution	Amount
CD4	RM4-5	BUV395	1:400	0.25 μL
F4/80	BM8	BV421	1:20	5 μL
CD11b	M1/70	BV510	1:400	0.25 μL
CD25	PC61	PerCP	1:100	1 μL
Ly6C	HK1.4	FITC	1:400	0.25 μL
CD45	30-F11	APC-H7	1:500	0.2 μL
FcR Block	145-2C11	N/A	1:100	1 μL
FACS Buffer	N/A	N/A	N/A	92.05 μL
Total	N/A	N/A	N/A	100 μL

Note: Mix for extracellular staining is prepared freshly and protected from light. Store at 4°C until use.

Mix for viability staining (100 μL per sample)

Antibody	Clone	Fluorochrome	Dilution	Amount
ViaKrome 808	N/A	IR885	1:100	1 μL
PBS	N/A	N/A	N/A	99 μL
Total	N/A	N/A	N/A	100 μL

Note: Mix for viability staining is prepared freshly and protected from light. Store at 4°C until use.

Mix for intracellular staining (100 μL per sample)

Antibody	Clone	Fluorochrome	Dilution	Amount
CD206	C068C2	BV650	1:30	3.33μL
FoxP3	3G3	PeCy7	1:100	1μL
Permeabilization buffer (ref. 00-8333-56)	N/A	N/A	1×	95.6μL
Total	N/A	N/A	N/A	100 μL

Note: Mix for intracellular staining is prepared freshly and protected from light. Store at 4 C until use. Discard any remaining volume and prepare a fresh solution for each experiment.

Step-by-step method details

CD4 T cell and monocyte isolation and differentiation into Treg cells and macrophages, respectively

Timing: 6 days (1 day for plate coating, 4 h for isolation protocols, 5 days for differentiation)

Here we describe the steps for isolating both monocytes and CD4+ T cells. These cells are then cultured for 5 days to allow differentiation into macrophages and regulatory T cells (Tregs), respectively (Figure 4A).

Figure 4.

Diagram representing the isolation of murine CD4+ T cells and monocytes and the establishment of co-culture conditions

(A) The scheme illustrates the isolation of murine bone marrow cells and splenocytes, followed by differentiation into M2-like macrophages and T regulatory cells, respectively.

(B) Gating strategy used to show the yield of the conversion into T regulatory cells.

(C) Gating strategy and yield obtained in the macrophage differentiation step.

Day−6.

• 1.
  Prepare the plate coating for the culture and differentiation of CD4+ T cells:

  • a.
    Add 100 μL of PBS supplemented with anti-CD3 (1 μg/mL) and anti-CD28 (0.5 μg/mL) per well of U-bottom 96-well culture plates.
  • b.
    Keep the plate in the incubator for 24 h.

Day−5.

• 2.Euthanize the mouse in sterile conditions.
• 3.Take the spleen and both hindlimbs of the mouse into 15 mL centrifuge tubes containing complete RPMI medium.
• 4.
  Isolate CD4+ T cells from spleen and differentiate them into T regulatory cells.

  • a.
    Use a 5 mL syringe piston to disrupt mechanically the spleen.
  • b.
    Pass the splenocytes through a 70 μm cell strainer.
  • c.
    Centrifuge at 480 rcf for 5 min.
  • d.
    Discard supernatant.
  • e.
    Lyse red blood cells with 3 mL of ACK lysing buffer for 5 min at room temperature.
  • f.
    Neutralize lysing solution with 10 mL of PBS.
  • g.
    Centrifuge at 480 rcf for 5 min.
  • h.
    Discard supernatant.
  • i.
    Resuspend the splenocytes in 5 mL of complete RPMI medium. Ensure a homogenous cell suspension.
  • j.
    Determine the number of cells preparing a 1:10 mix of cell suspension and Trypan Blue staining. Use a cell counter.

    Note: Collect a small amount of pre-isolated splenocytes to validate the efficiency in the differentiation process and perform flow cytometry (Figure 4B).

  • k.
    Isolate CD4+ T cells using the EasySep Mouse Naïve CD4+ T Cell Isolation Kit (Stemcell, #19765), according to the manufacturer’s instructions (https://cdn.stemcell.com/media/files/pis/10000003734-PIS_02.pdf).

    CRITICAL: Avoid pipetting thoroughly to increase the efficiency in the isolation process.

  • l.
    Prepare a homogenous cell suspension containing the isolated CD4+ T cells.
  • m.
    Determine the number of cells preparing a 1:1 mix of cell suspension and Trypan Blue and count the cells using a cell counter.

    Note: Since only a limited number of cells are expected, we recommend preparing a 1:1 mix to ensure a sufficient total cell count for accurate quantification.

  • n.
    Discard the PBS supplemented with anti-CD3 and anti-CD28 of the plate prepared the day before.
  • o.
    Wash the coated plate with 100 μL of PBS and discard it.

    CRITICAL: Add 50,000 isolated CD4+ T cells in 100 μL of CD4+ T cell culture medium. The yield of the differentiation process is close to 50%, so, we highly recommend stimulating a great excess of CD4+ T cells to have the proper number of fully differentiated T regulatory cells the day of seeding TImS (day 0 of the protocol).

  • p.
    Add 100 μL of (2×) Treg differentiation medium.
  • q.
    Allow the differentiation of CD4+ T cells for 5 days.
• 5.
  Isolate monocytes from bone marrow and differentiate them into M2-like macrophages.

  • a.
    Cut the bottom of a 0.2 mL PCR tube.
  • b.
    Cut both the upper and lower sections of the hindlimbs and bend the bones at the knee.
  • c.
    Insert the bone into the 0.2 mL PCR tube, allowing the bone marrow to pass through the opening of the PCR tube.
  • d.
    Place the 0.2 mL PCR tube containing the bone into a 1.5 mL microcentrifuge tube containing 300 μL of PBS (supplemented with 2.5% FBS).
  • e.
    Centrifuge at 3,000 rcf for 1 min at room temperature to pellet the bone marrow within the PBS (2.5% FBS).
  • f.
    Mix and pipette the bone marrow thoroughly to make a homogenous unicellular suspension.
  • g.
    Centrifuge at 480 rcf for 5 min at room temperature.
  • h.
    Carefully discard supernatant.
  • i.
    Lyse red blood cells with 3 mL of ACK lysing buffer for 5 min at room temperature.
  • j.
    Neutralize lysing solution with 10 mL of PBS.
  • k.
    Centrifuge at 480 rcf for 5 min.
  • l.
    Discard supernatant.
  • m.
    Resuspend the bone marrow in 5 mL of complete RPMI culture medium. Ensure a homogenous cell suspension.
  • n.
    Determine the number of cells preparing a 1:10 mix of cell suspension and Trypan Blue. Count with a cell counter.

    Note: Collect a small amount of pre-isolated bone marrow cells to validate the efficiency in the differentiation process and perform flow cytometry (Figure 4C).

  • o.
    Isolate monocytes using the EasySep Mouse Monocyte Isolation Kit (Stemcell, #19861), according to the manufacturer’s instructions. (https://cdn.stemcell.com/media/files/pis/10000005282-PIS_03.pdf).

    CRITICAL: Avoid pipetting thoroughly to increase the efficiency in the isolation process.

  • p.
    Prepare a homogenous cell suspension containing the isolated monocytes.
  • q.
    Determine the number of isolated monocytes preparing a 1:1 mix of cell suspension and Trypan Blue staining and count the cells.

    Note: Since only a limited number of cells are expected, we recommend preparing a 1:1 mix to ensure a sufficient total cell count for accurate quantification.

  • r.
    Add 10⁶ monocytes per well in a 6-well plate with 2 mL of Macrophage differentiation medium.
  • s.
    Place the plate into the incubator at 37°C and 5% CO₂.
  • t.
    Allow the macrophage differentiation process for 5 days.

    Note: Prepare the agarose molds 1–2 days before the TImS seeding (Day 0). Repeat the step 1 of the optimization for TSp section. The hydration of the mold must be done at least 2 h before seeding the cells inside the agarose molds.

Day−2.

• 6.
  Interleukins involved in the Treg differentiation are renewed to increase the yield of this step.

  • a.
    Carefully remove 100 μL of medium from the U-bottom 96-well plate with a multichannel pipette. Be careful to avoid aspirating cells from the bottom of the well.
  • b.
    Add 100 μL of (2×) Treg differentiation medium.

Day−1.

• 7.
  M2-like macrophage differentiation medium is added into the 6-well plate to allow polarization into M2-like phenotype.

  • a.
    Aspirate the medium from the plate where macrophages are differentiated.
  • b.
    Add 2 mL of M2-like macrophage differentiation medium.
  • c.
    Place into the incubator at 37°C and 5% CO₂.

Collect tumor and immune cells

Timing: 40 min

Here, we collect the three distinct cell populations (Tumor, Treg and Macrophages) and prepare the individual cell suspensions required for seeding TImS in the agarose molds.

Note: We recommend seeding an excessive number of CD4 T cells as the yield of Treg differentiation is approximately 50%. However, regarding macrophages, the whole amount of monocyte seeded are fully differentiated into M2-like macrophages.

Note: Steps 8, 9 and 10 are performed simultaneously the day that the TImS are seeded (Day 0 of protocol). In our case, the cell number selected for the TImS was determined to be 2,500. The ratio of cells is as follows: 10% Tumor: 50% T regulatory cells: 40% M2-like macrophages (10: 50: 40). This is equivalent to: 250 tumor cells, 1,250 T regulatory and 1,000 M2-like macrophages.

CRITICAL: We strongly recommend optimizing the ratio of seeded cells, as this is highly cell line dependent. In our protocol, the seeding ratio was independently optimized for each of the two cell lines used. However, as noted in “Problem 4”, this ratio may not be suitable for other cell types. For tumor cells with lower proliferative capacity, the seeding ratio may need to include a higher proportion of tumor cells. When making such adjustments, it is important to also account for the comparatively low proliferative potential of Tregs and M2-like macrophages in our experimental context.

Day 0.

• 8.
  Collect tumor cells.

  • a.
    Aspirate supernatant from flasks.
  • b.
    Add 5 mL of 0.05% Trypsin-EDTA and keep the cells in the incubator for 5 min (37°C and 5% CO₂).
  • c.
    Neutralize the trypsin with 5 mL of complete medium (complete RPMI for Lacun3 and complete DMEM for CMT167) and collect the cells into 15 mL centrifuge tubes.
  • d.
    Pellet the cells by centrifuging at 310 rcf for 5 min at room temperature.
  • e.
    Discard supernatant and re-suspend in 3 mL of the corresponding complete medium. Ensure a homogenous mix of cells.
  • f.
    Determine the number of cells preparing a 1:10 mix of cell suspension and Trypan Blue. Count the cells with a cell counter.
  • g.
    Keep the cells until use.
• 9.
  Collect T regulatory cells.

  CRITICAL: Collect the maximum number of T regulatory cells. We recommend to collect Treg cells directly from the U-bottom 96-well plate with a multichannel pipette to ensure enough number of cells needed in the following steps and transfer them into a 15 mL centrifuge tube.

  • a.
    Pellet the Treg cells by centrifuging them at 480 rcf for 5 min at room temperature.
  • b.
    Remove the supernatant as carefully as possible and leave 1 mL to avoid aspirating Treg cells.
  • c.
    Determine the number of cells preparing a 1:1 mix of cell suspension and Trypan blue. Count the Treg cells with a cell counter.

    Note: Since only a limited number of cells are expected, we recommend preparing a 1:1 mix to ensure a sufficient total cell count for accurate quantification.

  • d.
    Keep the cells until use.
• 10.
  Collect M2-like macrophages directly from the 6-well plate into a 15 mL centrifuge tube by scrapping them with a cell scrapper.

  Note: If the total volume is less than 2 mL, we recommend homogenizing the mix and directly using it to count the cells. If the volume is higher than 2 mL, we recommend centrifuging the cells (480 rcf for 5 min) and aspirating the supernatant while leaving 1 mL remaining.

  • a.
    Determine the number of cells preparing a 1:1 mix of cell suspension and Trypan Blue. Count the M2-like macrophages with a cell counter.

    Note: Since only a limited number of cells are expected, we recommend preparing a 1:1 mix to ensure a sufficient total cell count for accurate quantification.

  • b.
    Keep the cells until use.
• 11.
  Collect all the medium from each well of the 12-well plate into a 50 mL centrifuge tube. This medium can be used in the following steps.

  • a.
    Aspirate medium from the outside of the mold with a Pasteur pipette.
  • b.
    Carefully collect the medium from the inside of the mold with a 200 μL pipette from the corner of the agarose mold.

Prepare mixes and seed the TImS for viability assay

Timing: 1 h

In this section, we mix the three different cell populations that comprise the TImS. We recommend performing all necessary calculations in advance to minimize delays once the cells are ready for seeding. Prepare a seeding scheme for the plate. Here, we calculate the number of cells needed for a single replicate, both with and without radiotherapy (RT).

• 12.
  The plate is organized as follows.

  • a.
    Assay conditions:

    • i.
      TSp formed by Lacun3 cells without RT.
    • ii.
      TSp formed by CMT167 cells without RT.
    • iii.
      TImS formed by Lacun3 cells without RT.
    • iv.
      TImS formed by CMT167 cells without RT.
    • v.
      TSp formed by Lacun3 cells with RT.
    • vi.
      TSp formed by CMT167 with RT.
    • vii.
      TImS formed by Lacun3 cells with RT.
    • viii.
      TImS formed by CMT167 cells with RT.

CRITICAL: In this case, as an example, we calculate the number of cells for two molds per condition. To calculate the total number of cells needed for each mix, we consider 81 wells (9 × 9 well array) per mold and three molds per condition (accounting for 1 mold of excess). We prepare 4 different mixes per cell line.

• 13.
  Calculate the required number of cells.

  • a.
    For TSp: we make spheroids of 2,500 cell-size. This size was previously optimized.

    • i.
      $2,500cells\ast 81wells\ast 3molds=607,500cells$
  • b.
    For TImS, the final cell number is set to 2,500. We seed a ratio 10:50:40 of Tumor cells: Treg cells: M2-like macrophages, which corresponds with 250, 1,250 and 1,000 cells, respectively:

    • i.
      For Tumor cells: $250cells\ast 81wells\ast 3molds=60,750cells$
    • ii.
      For Treg cells: $1,250cells\ast 81wells\ast 3molds=303,750cells$
    • iii.
      For M2-like macrophages: $1000cells\ast 81wells\ast 3molds=243,000cells$
• 14.Prepare the 4 different mixes.

Note: In this case we need 190 μL/mold ∗ 3 molds = 570 μL per mix. If the volume of the mix upon adding the three different population is less than the required for each mix, we recommend to add the remaining volume of media up to 570 μL and homogenize the mix. If the volume is more than 570 μL, pellet the cells (480 rcf for 5 min), remove supernatant carefully and re-suspend up to the corresponding volume of medium.

• 15.Add 190 μL of mix per mold.
• 16.Keep the plate inside the incubator and wait for 30 min. Let the spheroids settle at the bottom of the molds.
• 17.Fill each well of the plate with 2 mL of the appropriate complete medium supplemented with IL2 (100 U/mL). The medium collected in step 11 can be directly supplemented with IL2 and used to fill up the wells.
• 18.Keep the plate in the incubator.

Preparation of the mixes and seeding the TImS for tracking experiment

Timing: 2 h

Here, we perform a tracking experiment to visualize the three distinct cell populations of the TImS. This step is optional. We can take this step concurrently with the previous protocol. Additionally, we need to include a preceding sub-step in which each cell population is labeled with its corresponding cell tracker prior to preparing the mixes. The calculations and sub-steps involved in preparing the mixes are the same as those described in steps 12 – 18 of the “prepare mixes and seed the TImS for viability assay” section.

• 19.Prepare a cell suspension of 10⁶ cells / mL for each cell population.

Note: We may adapt this cell suspension depending on the number of isolated cells and the number of cells we need to seed in the following steps.

• 20.Pellet the cells by centrifugation at 480 rcf for 5 min.
• 21.Remove supernatant carefully.
• 22.Re-suspend the cells in 1 mL of PBS.
• 23.
  Prepare stock solutions of cell trackers.

  • a.
    5 mM for Cell Trace Violet in DMSO.
  • b.
    5 mM for Cell Trace CFSE in DMSO.
  • c.
    1 mM for Cell Trace Far Red in DMSO.
• 24.
  Add 1 μL of each tracker stock solution to their corresponding cell suspension.

  • a.
    Cell Trace Violet → Tumor cells.
  • b.
    Cell Trace CFSE → T regulatory cells.
  • c.
    Cell Trace Far Red → M2-like macrophages.
• 25.Incubate the labeled cells in the incubator for 20 min.
• 26.Neutralize the dyes by adding 1 mL of complete medium.
• 27.Incubate the cells for 5 min inside the incubator.
• 28.Pellet the cells and resuspend in the desired volume of medium. Labeled cells are ready for preparing mixes and seeding in the agarose molds.

Note: We recommend counting the cells immediately after staining with cell trackers, as these dyes may be cytotoxic.

Note: To continue with the protocol, the steps are identical to those corresponding to steps 12-18 of the “prepare mixes and seed the TImS for viability assay” section.

Irradiation of the TImS in the molds

Timing: 20 min

Here, we irradiate the spheres using an X-ray beam linear accelerator (SARRP). In our experimental setup, cells are exposed to a dose of 6 Gy. The irradiation step is performed by qualified personnel.

Day 1.

• 29.One day after seeding the TImS, once the spheroids are compact, only the plates containing the molds designated for irradiation are carefully moved and irradiated using a SARRP irradiator.

CRITICAL: It is crucial to be extremely careful during the handling of the plate. In our case we have to move the cells from the incubator to the SARRP irradiator in the animal facility. We recommend to avoid sudden movements when carrying the plate to prevent displacements of the spheres. Upon irradiation, the plate is brought back inside the incubator.

TImS collection and staining with extra- and intracellular markers for flow cytometry analyses

Timing: 4 h

Here, we outline the steps used to assess the cell viability of TImS, with or without irradiation. Optimizing this protocol enables the study of how radiotherapy affects tumor cells in the presence of immunosuppressive cell populations (Figures 5A and 5B).

Figure 5.

Irradiation of TImS and analysis of cell death

(A) Example of the gating strategy showing the different populations included in the TImS: Tumor cells (CD45-); Non-converted CD4 T cells (CD45+CD4+CD25-FoxP3-), T regulatory cells (CD45+CD4+CD25+FoxP3+) and M2-like macrophages (CD45+CD11b+F4/80+CD206+). Viakrome 808 is used to stain non-viable cells.

(B) Graphical representation of the total number of immune cells (non-converted CD4+ T cells, T regulatory cells, and M2-like macrophages) present in each mold, with or without radiation, in both models studied. Data are expressed as mean ± SD and were analyzed with a student’s t-test. ∗∗∗: p <0.001.

Day 3.

• 30.
  Cell viability of the TImS is analyzed at day 3 of experiment.

  • a.
    Collect the TImS from the mold and place it into a 15 mL centrifuge tube (same as step 11 of the “optimization for TSp” section):

    • i.
      Remove 1 mL from the outside of the well.
    • ii.
      Collect the TImS along with the remaining medium by pipetting “up and down” several times within the agarose mold to ensure all TImS are gathered.
    • iii.
      Use PBS if needed to collect all the TImS of the mold. Repeat this process as necessary.
  • b.
    Centrifuge at 480 rcf for 5 min at room temperature.
  • c.
    Remove supernatant and wash the TImS with 2 mL of PBS.
  • d.
    Centrifuge at 480 rcf for 5min at room temperature.
  • e.
    Discard all the supernatant.
  • f.
    Use 300 μL of TrypLE Select 2× (prepared in PBS) to dissociate the TImS. Mix carefully.
  • g.
    Keep the TImS at 37°C for 20 min.

    Note: This incubation might be different depending on the cells included in the spheroids. It is recommended to test different concentrations of TrypLE and / or increase the incubation time.

    Note: The following steps are performed at 4°C. Place the PBS and FACS buffer on ice and refrigerate the centrifuge at 4°C.

  • h.
    Add 2 mL of cold PBS and mix vigorously.
  • i.
    Centrifuge at 480 rcf for 5 min at 4°C.
  • j.
    Discard PBS.
  • k.
    Resuspend in 200 μL of FACS buffer and transfer into a v-well plate.
  • l.
    Centrifuge at 480 g for 5 min at 4°C.
  • m.
    Discard supernatant
  • n.
    Perform extracellular staining as follows:

    • i.
      Add 100 μL of Mix for extracellular staining.
    • ii.
      Incubate 30 min at 4°C in darkness.
    • iii.
      Wash with 100 μL of FACS Buffer.
    • iv.
      Centrifuge at 480 rcf for 5 min at 4°C.
    • v.
      Discard supernatant
  • o.
    Viability staining:

    • i.
      Add 100 μL of Mix for viability staining.
    • ii.
      Incubate 30 min at 4°C in darkness.
    • iii.
      Wash with 100 μL of FACS Buffer.
    • iv.
      Centrifuge at 480 rcf for 5 min at 4°C.
    • v.
      Discard supernatant.
  • p.
    Intracellular staining:

    • i.
      Add 180 μL of Fixation/Permeabilization buffer (1/4 of concentrate Buffer (ref. 00-5123-43) + 3/4 of diluent Buffer (ref. 00-5223-56)).
    • ii.
      Incubate for 20 min at 4°C in darkness.
    • iii.
      Centrifuge at 480 g for 5 min at 4°C.
    • iv.
      Discard supernatant.
    • v.
      Wash twice with 200 μL of 1× permeabilization buffer (9/10 of distilled water + 1/10 of permeabilization buffer (ref. 00-8333-56)).
    • vi.
      Centrifuge at 480 rcf at 4°C in darkness.
    • vii.
      Incubate with 100 μL of Mix for intracellular staining for 30 min at 4°C in darkness.
    • viii.
      Wash with 100 μL of permeabilization buffer.
  • q.
    Centrifuge at 480 rcf for 5 min at 4°C.
  • r.
    Discard supernatant.
  • s.
    Resuspend cell in 200 μL of FACS Buffer.
  • t.
    Add 10 μL of Precision Count Beads (1000 beads/μL) and mix thoroughly.
  • u.
    Acquire samples in the CytoFlex LX or similar and analyze data with CytExpert software.

Visualization of tracked cells

Timing: 1 h

Here, we examine in detail the cell-cell interactions and the spatial distribution of each cell population. To achieve this, we image TImS labeled with different cell trackers. To assess treatment effects, we recommend performing this step on day 1, prior to radiotherapy (Figure 6) and on day 3 (post-RT).

• 31.Take bright-field images with Primovert microscope and Labscope app (ZEISS) (Figures 6A and 6B).

CRITICAL: Carefully transfer the agarose mold from the 12-well plate into a designated glass-bottom dish (GWST-5040) using tweezers. Visualize the TImS using a Confocal Laser Scanning Microscope (LSM800).

• 32.
  ZEN blue software (version 3.0) is used to process the images. Images of Lacun3 and CMT167, with or without radiotherapy, are captured at day 3 (Figures 6C and 6D).

  • a.
    Merged images are composed by:

    • i.
      Image from DAPI channel corresponds to tumor cells.
    • ii.
      Image from AF488 channel corresponds to T regulatory cells.
    • iii.
      Image from AF594 channel corresponds to M2-like macrophages.

Figure 6.

Irradiation of TSp and TImS and analysis of cell death

(A and B) Representative brightfield images of Lacun3- and CMT167-derived TSp and TImS with or without irradiation.

(C and D) Representative fluorescence images of Lacun3 TImS (C) and CMT167 TImS (D) at day 1 (untreated) and day 3 (after irradiation). Tumor cells are seen in blue (CellTrace Violet Cell Proliferation), M2-like macrophages in red (CellTrace Far Red Cell Proliferation) and T regulatory cells in green (CellTrace CFSE Cell Proliferation). Scale bar: 100 μm.

Expected outcomes

Using the present protocol, we have developed a novel scaffold-free 3D culture system⁵ with the capacity to address key questions in tumor immunology and serve as a platform for drug screening. By employing agarose molds, we provide cells with a soft, low-adherence matrix that promotes aggregation and spheroid formation, thereby supporting the development of TImS. This environment preserves 3D architecture and mimics native cell-cell interactions. For this model, we utilized two previously characterized murine lung adenocarcinoma cell lines, Lacun3 and CMT167.^1,2 We first optimized spheroid culture conditions by identifying the appropriate initial cell seeding density to align with experimental timelines. To determine this, viability, mitochondrial activity, and proliferation are evaluated in both models, concluding that 2,500 cells per spheroid is the optimal seeding number for our experiments. However, the protocol is highly versatile and can be adapted to other murine or human cancer cell lines with appropriate optimization of cell type and number.

Furthermore, our in vitro autologous 3D tumor-immune model closely reflects the immunosuppressive microenvironment of advanced lung tumors. To replicate this context, we co-cultured tumor cells with T regulatory cells (Tregs) and M2-like macrophages, creating a microenvironment that mimics immune suppression observed in vivo. Upon treatment—such as with radiotherapy—this model allows for a more physiologically relevant analysis of therapeutic responses.

Our results show that the addition of immune cells to the Lacun3 and CMT167 models creates a niche where tumor cells cluster centrally, with immune cells organizing around tumor cells, in the periphery. This distribution is something that can be frequently observed in Lacun3- and CMT167-derived tumors (not shown). The use of cell trackers facilitates detailed visualization of intercellular interactions. Viability assays further revealed that CD4+ T cells, including non-converted CD4+ T cells and Treg cells, are more sensitive to radiotherapy than M2-like macrophages.

In conclusion, this autologous, scaffold-free 3D culture system that incorporates both tumor and immunosuppressive immune cells, offers a cost-effective and physiologically relevant model to study the impact of radiotherapy. It provides a valuable platform for exploring cancer–immune cell interactions in the context of an immunosuppressive tumor microenvironment and evaluating responses to radiotherapy and immunochemotherapeutic agents.

Quantification and statistical analysis

The normality of data was assessed using the D’Agostino & Pearson test. Statistical differences between two groups were determined using Student’s t-test, while comparisons between more than two conditions were performed using one-way ANOVA. Results were represented as mean ± standard deviation (SD) of at least three independent experiments. Data were plotted and analyzed with GraphPad (GraphPad Prism 8.0.1), with statistical significance defined as p <0.05 (∗), p <0.01 (∗∗) and p <0.005 (∗∗∗).

Limitations

This protocol provides a detailed framework for studying interactions between tumor cells, T regulatory (Treg) cells, and M2-like macrophages. As previously noted, optimization steps are essential for generating TImS that reflect the cellular proportions typically found within a given tumor. While our system includes only two immune cell types, this limited composition does not fully capture the complexity of the tumor microenvironment (TME). Therefore, further research is necessary to develop more advanced co-culture systems that incorporate additional immune components and enable the evaluation of therapeutic agents.

A key challenge in these 3D co-culture models is the higher proliferative rate of tumor cells compared to immune cells. To address this, we adjust experimental time points and radiation treatment schedules to balance tumor cell growth with immune cell presence. These parameters require careful optimization based on the specific cell types, co-culture conditions, and treatment regimens used.

Another limitation of the current model is its exclusive use of murine cells. Although this approach is suitable for initial optimization of 3D culture conditions, translating these findings to human biology requires the development of human-derived TImS models. Future efforts should focus on establishing such human 3D co-cultures to more accurately replicate the TME and enhance the translational relevance of this platform.

Troubleshooting

Problem 1

Spheroids are lost before collecting them for performing viability or mitochondrial activity measurement protocols (Steps 11 and 12 of the optimization for TSp section).

Potential solution

• •Do not move the plate if it is not necessary to avoid sudden displacement of the spheroids.
• •Be careful when moving the plate. If needed, use a tray to support the plate.

Problem 2

Resazurin conversion values are extremely high in some wells (Step 12 of the optimization for TSp section).

Potential solution

• •Ensure the correct filters in the microplate reader.
• •During the seeding process, some cells might have attached to the well. Make sure that there are no cells attached to the well that may induce some false lectures.
• •Avoid making bubbles when pipetting the replicates in the plate.

Problem 3

Problems during the isolation processes. Number of Treg cells and/or M2-like macrophages is lower than needed for seeding the TImS (Steps 9 and 10 of the collect tumor and immune cells section).

Potential solution

• •Make sure the magnetic beads and reagents form the kit are correct.
• •During the isolation process, avoid mixing vigorously or “up-down” with the pipette, as you might lyse cells, thus reducing efficiency in the isolation step.
• •Differentiate all the isolated cells you get.
• •Ensure to add the proper amount of interleukins to increase the yield in the differentiation step.
• •Make sure you collect the maximum number of immune cells the day of co-culture.
• •Adjust the excess of cells to prepare the minimum volume of mix you need in your experiment.

Problem 4

Issues with the detection of immune cells at the end of the experiment (Steps 30, 31 and 32).

Potential solution

• •Ensure that the staining protocol is performed properly.
• •Adjust the ratio of tumor cells and immune cells. For high-rate proliferative tumor cells, the number of cells seeded may be much lower than that of immune cells.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Diego Serrano, dserrano@unav.es.

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Sergio León (sleon1@unav.es).

Materials availability

This study did not generate new unique reagents.

Data and code availability

This study did not generate datasets or codes.

Acknowledgments

We are grateful to Javier Garcia (Department of Pathology, Anatomy and Physiology, University of Navarra) and the Histology and Electron Microscopy (HEMS) platform at the i3S Institute for Research and Innovation in Health of the University of Porto for their technical help with the histological processing of the murine lung tumor and the murine lung TImS, as well as to the flow cytometry platforms at the i3S and CIMA centers. This work has been funded by a grant from the International Association for the Study of Lung Cancer (to D.S.), Instituto de Salud Carlos III (ISCIII)-FIS Pl23/01727 (to J.J.A.), Worldwide Cancer Research (grant #240390 to A.C.), Gobierno de Navarra-GRANATE (to A.C. and D.S.) and Gobierno de Navarra – Salud GN2024-07 (to D.S.), and “RADIORESISTANCE-Dissect the tumor microenvironment to battle cancer radioresistance and immune escape” (PTDC/MED-ONC/4165/2021). S.T.Q. (2022.11948.BD) acknowledges an FCT-funded PhD fellowship and the Doctoral Programme Biotech Health (ICBAS/FFUP), and F.C. acknowledges an FCT-funded CEEC contract (2021.01773.CEECIND). S.L.'s exchange in M.J.O.'s laboratory was funded by an EMBO Scientific Exchange Grant (#10721). D.S. is a recipient of a Ramón y Cajal grant (#RYC2022-038084-I) funded by MICIU/AEI/10.13039/501100011033 and “ESF+.”

Author contributions

Conceptualization, S.L., A.C., J.J.A., F.C., M.J.O., and D.S.; methodology, S.L., F.C., N.O., M.R., and D.S.; investigation, S.L., F.C., N.O., M.R., and D.S.; formal analysis, S.L.; writing – original draft, S.L., A.C., and D.S.; writing – review and editing, all authors; funding acquisition, A.C., J.J.A., F.C., and D.S.; supervision, A.C., M.J.O., and D.S. All authors read and approved the final version of the manuscript.

Declaration of interests

The authors declare no competing interests.