2.1 Patient PDAC tissue

All research involving human PDAC material obtained from patients participating in the Molecular Pancreas Program (MolPAC) at the University Medical Center of Göttingen, which includes the generation and utilization of PDAC PDOs, has received approval from local regulatory authorities (approval references: 11/5/17, 22/8/21Ü, and 2/4/19).

2.2 Murine tumor tissue

For mouse-derived PDAC tissue, 5 × 10^5 KPC cells were orthotopically implanted into the head of the pancreas of C57BL/6 J mice as described before [29]. Tumors developed within 2–3 weeks post-implantation and were excised during the section for organoid formation. KPC cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) with 10% Fetal Calf Serum (FCS, Gibco).

KPC cells were established from tumors of the KPC mouse that contains a conditional point mutation in the transformation-related protein TP53 gene (TP53R172H), and a point mutation in the KRAS gene (KRASG12D) both of which generate non-functional proteins [30]. The cells were kindly provided by Prof. Dr. Volker Ellenrieder (Klinik für Gastroenterologie, Gastrointestinale Onkologie und Endokrinologie, University Medical Center, Göttingen, Germany).

All animal procedures were performed in compliance with the guidelines of the ARRIVE, European Directive (2010/63/EU), and the German ethical laws and were approved by the administration of Lower Saxony, Germany (approval number G20.3527).

2.3 Medium preparation2.3.1 Digestion medium

Per 100 ml: 12 mg Collagenase type I (Sigma), 12 mg Dispase II (Sigma), and 1 ml of 10% FCS (Gibco) were added to 99 ml of DMEM (Gibco).

2.3.2 Human organoid growth medium (HOGM)

Per 50 ml of HOGM: 25 µl A83-01 (1 mM, Tocris), 50 µl Human Epidermal Growth Factor (hEGF; 500 µg/ml, Invitrogen), 50 µl human Fibroblast Growth Factor-10 (hFGF-10; 100 mg/ml, Peprotech), 50 µl Gastrin I (100 µM, Sigma), 125 µl N-acetylcysteine (500 mM, Sigma), 500 µl Nicotinamide (1 M, Sigma), 1 ml B-27 supplement (50x, Gibco), 100 µl Primocin (50 mg/ml, InvivoGen), 25 ml of Wnt3a-, 5 ml R-spondin and 50 µl of Noggin-conditioned media were diluted in 19 ml of organoid splitting medium (1x Glutamax (Gibco), 1x HEPES (Gibco), 1 ml 1x Primocin (InvivoGen), 30% Bovine Serum Albumin (BSA) (Sigma) (diluted in Advanced DMEM/F12 medium (AdDMEM/F12, Gibco). For the initial seeding, splitting, or thawing, 10 µM Y-27632 Rho Kinase Inhibitor (Sigma) was added to the organoid medium.

2.3.3 Murine organoid growth medium (MOGM)

Per 50 ml of MOGM: 5 µl murine Epidermal Growth Factor (mEGF; 500 µg/ml, Invitrogen), 50 µl murine Fibroblast Growth Factor-10 (mFGF-10; 100 µg/ml, Peprotech), 5 µl Gastrin I (100 µM, Sigma), 125 µl N-acetylcysteine (500 mM, Sigma), 500 µl Nicotinamide (1 M, Sigma), 1 ml B-27 supplement (50x, Gibco), 5 ml R-spondin and 5 ml of Noggin-conditioned media were diluted in 38.3 ml of organoid splitting medium (1x Glutamax (Gibco), 1x HEPES (Gibco), 1% P/S diluted in AdDMEM/F12 (Gibco). For the initial seeding, splitting, or thawing, 10 µM Y-27632 Rho Kinase Inhibitor (Sigma) was added to the organoid medium.

2.3.4 Wnt3a-, R-Spondin- and Noggin-conditioned media

For the preparation of Wnt3a-conditioned media, the L-Wnt3A cell line (ATCC® CRL-2647™) was acquired from ATCC and cultured in adherence to the guidelines provided by the manufacturer. A detailed protocol can be found in the work of Wilson et al. [31]. To generate R-spondin- and Noggin-conditioned media, 293T-HA-Rspol-Fc (obtained from Calvin Kuo’s group at Stanford University) and HEK293-mNoggin-Fc (sourced from AG Florian Greten and AG Herner Farin at CSH Frankfurt) cell lines were utilized. The preparation followed the protocol outlined by Klemke et al. [32]. Specifically, 293T-HA-Rspol-Fc or HEK293-mNoggin-Fc cells were thawed and seeded in 175 cm2 flasks with growth medium (comprising 500 ml DMEM (Gibco), 60 ml FCS, and 5 ml P/S) supplemented with 300 µg/ml Zeocin (Invitrogen) or 500 µg/ml Geneticin (Gibco), respectively. Upon reaching confluency, cells were expanded into eight (for 293T-HA-Rspol-Fc cells) or six (for HEK293-mNoggin-Fc cells) 175 cm2 flasks using antibiotic-free medium. To preserve the cells, one flask from each condition was aliquoted and supplemented with Zeocin or Geneticin before freezing. Subsequently, upon reaching confluency, the antibiotic-free medium was replaced with 50 ml of conditioning medium (AdDMEM/F12 medium supplemented with 1x Glutamax, 1 M HEPES (Invitrogen), and 1% P/S). After one week of culturing, the conditioned medium was collected, centrifuged (500 g, 4 °C, 5 min), pooled from all the flask supernatants, and then filter-sterilized using a 0.22 μm filter. The resulting R-spondin- or Noggin-conditioned media were collected into 15 ml conical tubes (5 ml per tube) and stored at -20 °C.

2.3.5 Organoid passaging medium (OPM)

Per 500 ml: 5 ml 100x Glutamax (Gibco), 5 ml 1 M HEPES (Gibco) and 1% P/S were added to 500 ml AdDMEM/F12 (Gibco).

2.3.6 PBMCs culture medium

RPMI medium (Gibco) supplemented with 10% FCS, 50 µM β-mercaptoethanol (Gibco), and 1% P/S.

2.4 PDAC organoid establishment and culturing

Two human organoid cultures were established from primary tumors obtained from two independent PDAC patients (described on Sect. 2.1) during surgical intervention, designated Human Organoid 1 (HO1) and Human Organoid 2 (HO2). Human PDAC organoids (PDAC PDOs) were generated from PDAC specimens following standard organoid cell culture methods established as described before [33]. PDAC tissue was extracted from the tumor bulk following resection and confirmation of a PDAC diagnosis. Additionally, Murine Organoid 1 (MO1) and Murine Organoid 2 (MO2) were derived from tumor tissue obtained from two different KPC-tumor-bearing C57BL/6J mice (described in Sect. 2.2). Generation and culturing of both murine and human organoids followed the Tuveson Laboratory Murine and Human Organoids Protocols, accessible at http://tuvesonlab.labsites.cshl.edu/wpcontent/uploads/sites/49/2018/06/20170523_OrganoidProtocols.pdf. In brief, freshly excised tumor tissue was cut into small fragments and resuspended in the digestion medium for enzymatic digestion. After obtaining a cell suspension, a 50 µl mixture of suspended cells with Growth Factor Reduced (GFR) Matrigel (Corning) was added to a preheated 24-well plate. Following the solidification of the dome through a 15-min incubation, the respective organoid growth medium for MOGM or HOGM was applied to the top of the dome. Human and mouse organoid formation was microscopically observed after 3 days of culturing at 37 °C under a humidified atmosphere of 5% CO2. The organoids were utilized for experimentation after at least 3 passages.

2.5 Chemotherapy treatment of organoids

On day zero, fully matured organoids were harvested and subjected to mechanical dissociation using a 200 µl tip affixed to a 10 ml serological pipette. A single-cell suspension was subsequently obtained by incubating the organoids for 3 min with Trypsin/EDTA (0.25%/0.02% (w/v) in PBS) solution at 37 °C. Following this, a cell density of 1 × 10^4 cells was mixed with 15 µl of Matrigel and plated in 48-well plates using the murine (MOGM) or human organoid medium (HOGM), as outlined in the protocol available at http://tuvesonlab.labsites.cshl.edu/wpcontent/uploads/sites/49/2018/06/20170523_OrganoidProtocols.pdf, with Y-27632 supplementation.

After 3 days, serial dilutions of gemcitabine (Gemcitabine Hexal(R), 40 mg/ml; ranging from 3 nM to 100 nM) were prepared in murine or human organoid medium and added to the matured organoids. Live imaging to observe the effects of gemcitabine on PDAC organoids was performed for 4 days, after which endpoint ATP levels were measured using the CellTiter-Glo(R) 2.0 reagent (Promega).

2.6 Human and murine PBMC isolation and pre-activation

Blood was collected from healthy C57BL/6J mice via cardiac puncture following euthanasia. Human cells were isolated from Leukocyte Reduction System (LRS) chambers of the local blood bank (UMG ethics approval 29/07/23). After LRS processing, PBMCs of healthy human thrombocytes from HLA-A*02:01 donors and of healthy mice were isolated by density gradient centrifugation utilizing SepmateTM tubes (Stemcell Technologies, 85450 for human and Stemcell Technologies, 85415 for mouse samples) containing Lymphocyte Separation Media (1,077 g/ml, Anprotec), following the manufacturer’s instructions.

The resulting PBMCs were counted and seeded at a density of 1 × 10^5 cells per well in a 96-well round bottom plate in PBMCs medium (RPMI medium (Gibco), supplemented with 10% FCS, 50 µM β-mercaptoethanol (Gibco), and 1% P/S). Following an overnight incubation, murine PBMCs were pre-activated by suspension in LymphoGrow II medium (Cytogen), while human PBMCs were pre-activated by adding 10 µl of ImmunoCult™ Human CD3/CD28 T cell Activator (StemCellTechnologies) per 1 ml of PBMCs medium. After a 24 h pre-activation period, both human and murine PBMCs were used for human and mouse organoid co-cultures, respectively.

2.7 Human- and mouse-derived pancreatic cancer/immune cell co-cultures

Murine and human organoids were prepared as described in Sect. 2.4. On day 3, matured organoids were carefully collected using a 200 µl tip (tip end cut) attached to a 10 ml serological pipette and resuspended in organoid passaging medium to remove the Matrigel. After 3-min centrifugation at 300 g, the medium was removed, and 20 µl of the passaging medium containing pre-activated PBMCs (2.6) was added.

To prepare the plate for co-culture, 30 µl of 25% Matrigel (diluted in DMEM 1x) was added to each well of a 96-well plate. After solidification of the Matrigel through a 15-minute incubation at 37 °C, 20 µl of the PBMCs-organoids mixture was added on top of the first Matrigel layer. Subsequently, an equivalent concentration of Matrigel was added on top of the mixture. Following this, 200 µl of a 50% mixture of MOGM or HOGM and 50% PBMCs culture medium was added on top of the solidified Matrigel layer. For anti-PD-LI treatment, 0.1 mg/ml Atezolizumab (Atezolizumab Tecentriq(R) 1200 mg/20 ml) was added to the 50% MOGM or HOGM/ 50% PBMCs medium.

2.8 Live cell imaging

The growth and viability of organoids and organoid co-cultures were monitored with the live-cell imaging system IncucyteR S3 (Sartorius, Germany). Phase-contrast images were acquired every 4 h up to 100 h using the Organoid mode.

2.9 OrganoIDNet analysis

Following image acquisition with the Incucyte imaging system, bright field (BF) images were analyzed using an in-house developed deep-learning-based analysis software called OragnoIDNet. Briefly, we trained a StarDist model [34] on a custom dataset consisting of manually annotated BF images from the Incucyte imaging system. This dataset featured both human and mouse PDAC organoids, with and without PBMCs. This model was then employed to segment the organoids. Post-segmentation, organoids were categorized based on a mean pixel intensity threshold as either healthy or unhealthy. Additionally, for each condition, the combined areas of all segmented organoids were computed and then size-categorized into five bins: Tiny, Small, Medium, Large, and Huge. Moreover, the deviation of the organoid shape from a perfect circle was measured as Eccentricity, with a value of zero denoting a perfect circle. The analysis output from OrganoIDNet was further correlated with the current organoid analysis standard assay, CellTiter-Glo assay (for drug screening). The OrganoIDNet image analysis software we developed is available to researchers upon reasonable request and discussion with the corresponding author.

2.10 CellTiter-Glo analysis

Cell viability was assessed utilizing the CellTiter-Glo assay (Promega). Following stimulation with either chemotherapeutic agents or Atezolizumab monitored by live cell imaging, 80 µl of CellTiter-Glo reagent was added to each well of a 96-well plate. Subsequently, the plate was mixed for 2 min, followed by a 10 min incubation at room temperature (RT) in the dark to stabilize the luminescence signal. The acquired luminescence signal was then recorded using the multimode plate reader CLARIOstar (BMG LABTECH, Germany) and analyzed by MikroWin 2000 lite Version 4.43.

2.11 Immunofluorescence staining of organoids

Murine PDAC organoids were stained according to the protocol by Yan et al. [35]. First, PDAC organoids were gently separated from the Matrigel matrix using cold PBS/0.1% BSA (PBSB). The absence of Matrigel was confirmed by visual inspection after incubating the suspension in PBS on ice. The organoids were then fixed in 4% paraformaldehyde (PFA) for 30 min. Fixative was removed by rinsing with PBS, and the whole-fixed organoids were stored in PBS at 4 °C until immunofluorescence staining.

Organoids were blocked using PBSDT (PBS supplemented with 0.5% Triton X-100) to minimize nonspecific binding. The following primary rabbit antibodies were used: a polyclonal anti-SRY-Box Transcription Factor 9 (SOX9; Sigma,1:600, ab5535) and a monoclonal anti-Cytokeratin 19 (CK19; Abcam,1:200). The antibodies were diluted in fresh PBSDT solution and applied to the organoids for incubation at 4 °C for 48 h. Organoids were subsequently washed with PBSB (0.1% BSA in PBS) and secondary anti-rabbit antibody Alexa Fluor 546 (Invitrogen, 1:200) was added and incubated for 2 h. Following further 2 washing steps, Hoechst 33342 fluorescent dye (Invitrogen, 1:5000) was added for 15 min at RT for nuclear staining. Finally, the organoids were mounted on glass slides using Acqua-Poly/Mount (Polysciences) mounting media. Imaging was performed using a Zeiss LSM880 confocal laser scanning microscope (CLSM, Carl Zeiss Microscopy GmbH) with a 40x oil objective lens. Alexa Fluor 546 was excited at 561 nm, and Hoechst dye was excited at 405 nm. Single plane and 3D images from microscopy Z-stacks were processed and analyzed using IMARIS 9.1.2 software.

2.12 Flow cytometry analysis of PDAC organoids before and after co-cultures

For flow cytometric phenotyping, the cells were resuspended in 1x PBS/0.5% BSA/2 mM EDTA (PEB) buffer. Up to 10^6 cells were resuspended in 100 µl per staining, transferred to a 96-well plate, and centrifuged at 300 g for 5 min. The pellets were resuspended in 35 µl PEB buffer and 5 µl human FcR Blocking Reagent (Miltenyi Biotec,130-059-901) was added to each well. The sample was mixed, followed by the addition of the different fluorescent labeled primary antibodies for staining (1 µl each; see Table 1), mixed again and incubated for 10 min at 4 °C. Unbound antibodies were removed by washing twice with 250 µl of PEB. The pellets were then resuspended in a 150 µl PEB buffer. For identification of dead cells, propidium iodide (1 µg/ml) was added immediately before sample acquisition. Data was acquired on a MACSQuant Analyzer 16 and data analysis was performed with MACSQuantifyTM software (both Miltenyi Biotec). Gating strategy for PD-L1 expression from immune cell infiltration into human organoids in co-cultures is shown in supplementary Fig. S3, and from non-immune cells in supplementary Fig. S4.

Table 1 List of antibodies (Miltenyi Biotec) used for flow cytometric analysis2.13 Statistical analysis

Statistical analysis was performed with GraphPad Prism 9. All presented data are expressed as means with corresponding standard errors of the means (SEMs). Biological samples were mean ± SEM of technical replicates. Comprehensive information regarding the significance tests, including the number of replicates, and associated p values can be found in the respective figure legends.