Analysis of EDB-FN1 expression in spheroids models of pancreatic cancer

Initially, we validated the chemical features of the drug. Supplementary Fig. 1a shows the molecular structure of L19-IL2, a clinical-stage immunocytokine [39]. The non-covalent homodimer runs as a monomer in SDS-Page (Supplementary Fig. 1b) with a molecular size of ~ 42 kDa, while the SEC profile (Supplementary Fig. 1c) shows the dimeric product (~ 84 kDa). We confirmed the biological activity of L19-IL2 through a proliferation assay on CTLL2 cells (Supplementary Fig. 1d). The fusion protein exhibited IL-2 activity that closely matched the previously reported results for this product [30, 40, 41]. The 3D cultures better recapitulate the complexity of the TME and the interactions between cancer cells and stromal components [42]. First of all, we conducted IHC analysis to assess the expression of the stromal component, specifically of fibronectin, in spheroid models (13KC, KPC416, KPC06 and KPC12) (Fig. 1a). Afterwards, we evaluated the expression of EDB-FN1 using RT-PCR analysis (Fig. 1b) and determined the ratio of exclusion and inclusion of the alternative splicing isoform relative to the expression of total fibronectin (Fig. 1c). The results demonstrated that our models not only express EDB-FN1, but also exhibit an elevated isoform expression ratio compared to total fibronectin. Additionally, we performed IF analysis to assess the L19 ability to specifically recognize EDB-FN1 expressed in our spheroids (Fig. 1d). Among the available models, we selected KPC06 (Low immunogenic) and KPC12 (Non immunogenic) cancer cells, which closely mimic human pancreatic tumors. Indeed, these models do not elicit a robust immune response and, as previously shown, are resistant to immunotherapy [32, 43].These analyses collectively confirm that immunosuppressive KPC06 and KPC12 express EDB-FN1 and are suitable models for testing the L19-targeted antibody.

Fig.1

Characterization of the expression of EDB-FN1 in mouse pancreatic cancer models. a Histochemical analysis of Fibronectin expression in different 3D models of PDAC. 10X images of Hematoxylin/Eosin, 40X images of Fibronectin. Images shown are representative of 1 out of more than 10 fields acquired. b Real-time analysis of EDB-FN1 in spheroid models of PDAC. c Analysis of inclusion splice junction (pink bars) and exclusion splice junction (light blue bars) reads of EDB-FN1. d IF analysis on spheroids confirmed L19 was able to recognize EDB-FN1. Protein analyzed (in red) and nuclei (in blue) were reported. Images shown are representative of 1 out of more than 10 fields acquired. Bar plot showing the fold increase in EDB-FN1 fluorescence calculated as the ratio between the mean of CTCF quantified in each group and the mean of CTR. P-value < 0.05 was indicated in figures with one asterisk (*), P-value < 0.01 with two asterisks (**), P-value < 0.001 with three asterisks (***) and P-value < 0.0001 with four asterisks (****)

Establishment of an ex-vivo immunity-spheroid interaction platform

To investigate L19-IL2 effects in a complex system that recapitulates the main tumor and immune cell components, we developed an immune-spheroid interaction platform with KPC06 and KPC12 and tumor-antigen cytotoxic T-lymphocytes (CTLs), as described in Agostini et al. [31]. Briefly, PDAC expresses several tumor-associated antigens (TAAs), and among them, TERT has been extensively studied for immunotherapy [40, 41]. In line with these premises, we used mouse TERT specific T-lymphocytes [42] cultured as described by De Sanctis et al. [43]. To evaluate the effect of L19-IL2 on the activity of T-lymphocytes in the recognition of cancer cells we treated KPC06 and KPC12 with L19-IL2 for 2 h. After the treatment, TERT specific T-lymphocytes [34] labelled with CellTracker Red CMPTX Dye were added to the platform. The spheroids were previously labeled with CellEvent Caspase-3/7 Green ReadyProbes and analyzed by time-lapse live microscopy to measure apoptosis induction.

As expected, T-lymphocytes were able to recognize and engage with the cancer cells, triggering a significant increase in apoptotic cell death. However, treatment with L19-IL2 resulted in a marked enhancement of the cytotoxic activity of T-lymphocytes. This augmented killing capacity led to a pronounced increase in apoptosis specifically in the KPC06 and KPC12 tumor cell lines (Fig. 2a).

Fig. 2

Evaluation of the L19-IL2 effect on ex-vivo interaction platforms. a Immunity-spheroid interaction platforms with TERT specific T-lymphocytes and KPC06 and KPC12 treated with L19-IL2. The induction of apoptosis was evaluated using the CellEvent Caspase-3/7 Detection Reagent (green), while T-lymphocytes were stained with the vital staining CellTracker Red CMPTX Dye (Red). The platforms were monitored daily, and fluorescence images were acquired using the EVOS FL Auto 2 Cell Imaging System over a 48 h period. Images shown are representative of 1 out of more than 10 fields acquired. b Bar plot showing the fold increase in Caspase 3/7 activity in comparison to CTR. The fold increase is calculated as the ratio between the mean of CTCF quantified in each group and the mean of CTR. P-value < 0.05 was indicated in figures with one asterisk (*), P-value < 0.01 with two asterisks (**), P-value < 0.001 with three asterisks (***) and P-value < 0.0001 with four asterisks (****)

The impact of L19-IL2 on T-lymphocytes recruitment was found to be consistent across the two 3D models assessed, KPC06 (Low immunogenic model) and KPC12 (Non immunogenic model) (Fig. 2b).

Overall, these findings suggest that L19-IL2 can increase T-lymphocytes infiltration and antitumor activity in 3D pancreatic tumor models.

The L19-targeted antibody specifically hits EDB-FN1 of mouse and human cancer tissues

We assessed the expression of EDB-FN1 in tumor tissues derived from our PDAC models by both RNA-seq (Fig. 3a) and IF (Fig. 3b) using the L19 antibody, while the KSF antibody (specific to hen-egg lysozyme) was used as negative control. Moreover, we assessed the EDB-FN1 expression pattern also in PDAC patient samples (Supplementary Fig. 2).

Fig. 3

The L19-targeted antibody specifically hits EDB-FN1 of mouse cancer tissues. a Graphic representation of inclusion splice junction (green bars) and exclusion splice junction (blue bars) reads of EDB-FN1 from RNA-seq raw data in PDAC model tissues. b L19-targeted antibody in IgG1 format specifically target EDB-FN1 (Green) in vivo mice tumor tissues, while KSF antibody (specific for hen egg lysozyme, an irrelevant antigen) was used as negative CTR. c IF-based biodistribution analysis in orthotopic KPC06 mice. EDB-FN1 shown in green, saline was used as negative CTR

We performed an IF-based biodistribution analysis in mice bearing KPC06 tumors. L19-IL2 showed a preferential accumulation in tumors 24 h after intravenous administration. No uptake could be detected in healthy organs or in animals injected with saline solution (Fig. 3c).

These results suggest that L19-IL2 has the potential to be an effective targeted treatment for pancreatic tumors, with high selectivity for tumor cells over normal tissues.

In vivo characterization of immunocytokines sensitivity in syngeneic orthotopic mouse PDAC models

To test the effectiveness of different immunocytokines in pancreatic tumor models, C57BL/6J mice (RRID:IMSR_JAX:000664) were orthotopically injected with KPC06 and randomly assigned to receive once a week for 2 weeks: vehicle, as CTR, L19-IL2 high dose (100 µg/mouse), L19mIL12 (12 µg/mouse), mIL2-F8-mTNF(mut) (40 µg/mouse), L19mTNF (4 µg/mouse), standard chemotherapy with gemcitabine 10 mg/kg + abraxane 3 mg/kg (Gem/Abx) (Supplementary Fig. 3).

As expected, standard chemotherapy did not prove effective in reducing tumor volume when compared to the CTR group. As for the different immunocytokines, except for L19mTNF which failed to lead to positive results, the others (L19-IL2, L19mIL12, and mIL2-F8-mTNF(mut)) demonstrated almost complete tumor elimination (Supplementary Fig. 3a).

Additionally, the impact of these immunocytokines on the median survival rate was evaluated (Supplementary Fig. 3b). We observed that standard therapy (Gem/Abx) and L19mTNF failed to prolong mice median survival rate. On the contrary, L19-IL2, L19mIL12, and mIL2-F8-mTNF(mut) were able to cure all tumor-bearing mice. They were ultimately sacrificed when tumor volume reached the cut-off. To assess the possible adverse effects of the different immunocytokines, we measured changes in body weight, thus finding no substantial weight loss (Supplementary Fig. 3c). In summary we tested various immunocytokines in pancreatic tumor models, finding that, unlike standard chemotherapy, L19-IL2, L19mIL12, and mIL2-F8-mTNF(mut) effectively eliminated tumors and extended survival, while L19mTNF and standard therapy did not improve outcomes.

Dose-dependent reduction of tumor volume in syngeneic orthotopic mouse PDAC models following L19-IL2 treatment

Based on the previous results, we chose to focus on L19-IL2 for several reasons. PDAC are notoriously resistant to standard therapies and often display poor T-lymphocyte activation and limited recruitment of natural killer (NK) cells. Therefore, the ability to selectively target EDB-FN1 within the TME and locally increase IL-2 concentrations—thereby enhancing the activation and proliferation of effector cells—could be a key strategy for effective tumor eradication. The importance to selectively deliver IL-2 to the site of disease using the L19 antibody has been previously demonstrated in multiple murine tumor models [39, 44,45,46]. Moreover, based on the promising results obtained with the high dose of L19-IL2 (100 µg/mouse) in KPC06 mice, we decided to test a lower dose (30 µg/mouse) to evaluate its impact on tumor volume reduction. Our findings revealed that L19-IL2 low-dose resulted in a smaller, yet still significant reduction in tumor volume growth when compared to L19-IL2 high-dose (Supplementary Fig. 4).

L19-IL2 potentiates the activity of FOLFOX in syngeneic orthotopic mouse PDAC models

To evaluate the effects of L19-IL2 in combination with standard therapy (FOLFOX) on tumor volume and survival rates of PDAC models with different immunogenic potential, C57BL/6J mice (RRID:IMSR_JAX:0006649) were orthotopically injected with KPC06 (Low immunogenic model) and KPC12 (Non immunogenic model). In order to assess the effects of the combination, different dosage levels of the immunocytokine were selected: L19-IL2 low dose (30 µg/mouse) for KPC06 and L19-IL2 high dose (100 µg/mouse) for KPC12.

We selected a low dose for KPC06 based on the results from the dose–response experiment, while a high dose was chosen for KPC12 due to its characteristics as a non-immunogenic and more aggressive model with lower EDB-FN1 expression.

The mice were randomly divided into 4 groups (n = 8 mice in each group). The groups were treated with standard therapy (FOLFOX i.p., once a week for two weeks), or vehicle, as CTR, and L19-IL2 low dose for KPC06 and a high dose for KPC12 (i.v., 2 injections with 5-day interval) alone or in combination with FOLFOX. Treatments started when tumor volume reached ~ 50mm3. To evaluate the potential side effects of the treatment, body weight loss was assessed in both low and non immunogenic models (Fig. 4c and Supplementary Fig. 5c). No significant weight loss occurred in any treatment group, thus indicating the safety of the therapy.

Fig.4

L19-IL2 treatment effects in combination with FOLFOX in KPC06 model. Plot showing tumor growth curves of KPC06 tumor-bearing mice randomly assigned to receive vehicle, as CTR, standard therapy (FOLFOX i.p., once a week for 2 weeks), and L19-IL2 (30 µg/mouse i.v., once a week for 2 weeks) alone or in combination with FOLFOX; b Kaplan–Meier survival analysis of KPC06 mice, grouped according to each experimental condition. c Variation of body weight in the different treatment groups

L19-IL2 as a single agent showed a reduction in tumor growth that was comparable to that obtained with FOLFOX treatment. Additionally, when combined with FOLFOX, L19-IL2 led to a statistically significant decrease in tumor growth compared to the other treatments administered alone (Fig. 4a). The effect of L19-IL2, FOLFOX and combination therapy on median survival rate was also evaluated (Fig. 4b). Our findings revealed that FOLFOX (28 days vs 29 days; Chi square = 2.064; df = 1; P-value = 0.1508) and L19-IL2 treatment (28 days vs 30 days; Chi square = 3.493; df = 1; P-value = 0.0616), when used as single agents, were unable to extend mouse median survival. However, the combination treatment proved to be more effective than the individual treatments and significantly prolonged mouse median survival (28 days vs 33 days, Chi square = 9.151; df = 1; P-value = 0.0025).

Overall, here we demonstrated that L19-IL2 alone reduced tumor growth similarly to FOLFOX, but when combined with FOLFOX, it significantly decreased tumor growth and extended median survival compared to either treatment alone.

The KPC12 model presented no statistically significant reduction in tumor growth in any treatment group at day 18 (Supplementary Fig. 5a). Afterwards we were unable to verify the effects on tumor volume as the mice in the control and FOLFOX groups died before the others, so measurement data were not available.

We observed that L19-IL2 resulted in a significant increase in survival, both as a single agent (22 days vs 30 days, Chi square = 5.552; df = 1; P-value = 0.0185) and in combination (22 days vs 39 days, Chi square = 5.552; df = 1; P-value = 0.0185). While, as well as in the low immunogenic model, no significant differences were observed compared to FOLFOX (22 days vs 19 days, Chi square = 0.9724; df = 1; P-value = 0.3241) (Supplementary Fig. 5b).

L19-IL2 increases immune infiltrate into the tumor core

Tumor bulks (n = 3) from KPC06 mice treated with FOLFOX, L19-IL2, and the combination of both agents were characterized by 3’mRNA-seq to unravel the effects of L19-IL2 on pancreatic tumors (Fig. 5a). The analysis clearly showed that there was a consistent effect of L19-IL2 alone or in combination with FOLFOX on immune activation, and cytotoxic activity. A total of 117 and 101 genes were upregulated in L19-IL2 and L19-IL2 + FOLFOX treated mice respectively in contrast to CTRs (Fig. 5b and c). Among these we found a considerable over regulation of IL-2 receptors Il2Ra (CD25) and Il2Rb (CD122) and cytotoxic-related genes (Fig. 5e) highlighting the effective immune activating function of L19-IL2. On the contrary, among the 158 genes upregulated in FOLFOX mouse there was a decrease in cytotoxicity genes (Fig. 5d). In fact, a consistent increase of expression of immune response and T-lymphocyte activation signatures were found in the comparison between L19-IL2 and FOLFOX (Supplementary Fig. 6) and L19-IL2 plus FOLFOX and FOLFOX alone (Supplementary Fig. 7).

Fig. 5

Differential expression analysis on KPC06 treated mice show immune response activation. a Plot showing Principal Component Analysis on RNA-seq data. b Volcano plot showing the genes differentially expressed (log2 Fold Change ≦ −1.5 ≧ 1.5, FDR < 0.05) in the comparison between L19-IL2 treated mice and control (CTR). c Volcano plot showing the genes differentially expressed (log2 Fold Change ≦ −1.5 ≧ 1.5, FDR < 0.05) between mice treated with a combination of L19-IL2 and FOLFOX and CTR. d Volcano plot showing the genes differentially expressed (log2 Fold Change ≦ −1.5 ≧ 1.5, FDR < 0.05) between FOLFOX treated mice and CTR. e Bar plot showing log2 Fold Change value for T-lymphocyte activation genes resulting from DEA between the different treatment groups

To further validate these findings, we analyzed selected tumor tissue regions with Stereo-Seq OMNI ST technology. With this technology, we were able to characterize the cell population heterogeneity associated with the effect of L19-IL2 and FOLFOX with a resolution of 50 μm. We identified a total of 22 different clusters annotated according to the main cell type identified using SingleR (Fig. 6a and b). We found that L19-IL2 enhances immune infiltration in concomitance of FN1 expression, similarly to what we have found with bulk 3’mRNA-seq. The administration of L19-IL2 in combination with FOLFOX or as single agent had a potent effect on recruitment and activation of both CD8a+ T-lymphocytes and NK into the tumor front (Fig. 6c and d; Supplementary Fig. 8b-d), while those cells where not present in both CTR and FOLFOX treated tumors.

Fig. 6

Stereo-Seq OMNI spatial transcriptomics analysis. a,b Spatial clustering of Stereo-seq OMNI data on the four cores analyzed. c Spatially resolved clusters of L19-IL2 + FOLFOX sample and heatmap showing an enhanced inflammation in the tumor core with a major representation of Cd8a+ Activated T-lymphocytes, Cd74+ H2-D1+ APCs and Ikzf3+ NK. d Spatially resolved clusters of L19-IL2 sample and heatmap showing an inflamed tumor core

The presence of such cells localized with the high expression levels of both Il2Ra and Il2Rb (Supplementary Fig. 8a), two IL-2 receptors that orchestrate activation of both T-lymphocytes and NK, entailing the attractant and activating role of L19-IL2 role in tumors [39, 44, 45]. Moreover, ST showed how that L19-IL12 induced a consistent increase of Antigen-presenting cells (APCs) expressing MHC-II genes (Cd74, H2-Eb1, H2-Ab1), MHC-I genes (H2-D1, H2-K1) and co-stimulatory CD80 and CD86 potentiating T-lymphocytes activation signaling (Fig. 6c and d, Supplementary Fig. 8e).

By ST we demonstrated that KPC06 tumor bearing mice treated with L19-IL2 alone, or especially when combined with FOLFOX, significantly enhanced immune activation and cytotoxicity, evidenced by upregulation of key immune-related genes and increased infiltration of CD8a+ T-lymphocytes and NK cells into the tumors, while FOLFOX alone mainly boosted cytotoxicity genes. To validate these findings, we performed IF analysis on FFPE tumor samples of KPC06 (Fig. 7) and KPC12 models (Supplementary Fig. 9). Specifically, we aimed to verify the expression of CD8+ TILs, GRZB+ cytotoxic effector cells, and PRF1+ cytotoxic effector cells across the different treatment groups confirming the major effect on T-lymphocyte activation of L19-IL2 plus FOLFOX therapy.

Fig. 7

Immunofluorescence analysis on KPC06 models. IF analysis for CD8.+ TILs, GRZB and PRF1 in KPC06 tumor tissues. Protein analyzed (in green) and nuclei (in blue) are reported. Images shown are representative of 1 out of more than 10 fields acquired. Bar plots show percentage of positive cells grouped by treatments. P-value < 0.05 was indicated in figures with one asterisk (*), P-value < 0.01 with two asterisks (**), P-value < 0.001 with three asterisks (***) and P-value < 0.0001 with four asterisks (****)

We demonstrated that L19-IL2 affects tumor growth and immune cell infiltration in KPC06 tumors using RNA-seq, ST, and IF. L19-IL2 significantly increased immune cell infiltration, particularly CD8+ T-lymphocytes and cytotoxic cells, and enhanced the efficacy of FOLFOX. The combination treatment showed the greatest increase in immune cells, while L19-IL2 alone had a smaller effect in the less immunogenic KPC12 model.