Triggering anti-GBM immune response with EGFR-mediated photoimmunotherapy

The conjugation of IRDye700DX-maleimide (IR700, ex. 689 nm, em. 700 nm; LI-COR® Bioscience, USA) to the Z_EGFR:03115-Cys affibody molecules (Affibody, Sweden) is described in detail in the supporting information (Additional File 1).

Human GBM cell line DKMG and murine GBM cell line GL261 were purchased from the Celther Polska (Poland) and the German Collection of Microorganisms and Cell Cultures (DSMZ, Germany), respectively. U87-MG and U87-MGvIII were kindly provided by Dr. Frank Furnari (Ludwig Cancer Research, USA) [23]. The primary, patient-derived cell lines WSz4, WSz50, and WSz57 have been recently established in our lab [22]. The cells were grown as described in the supporting information. BL6-NPE-GFP-Luc murine GBM cell line was kindly provided by Dr. Steven Pollard (University of Edinburgh, UK) and cultured as previously reported [24]. The genetic origin of all the cell lines was tested and authenticated by short tandem repeat (STR) DNA profiling analysis (Eurofins Medigenomix, Germany). The cells were also routinely tested and found to be negative for Mycoplasma contamination (PCR detection kit, Surrey Diagnostics Ltd., UK).

Singlet oxygen (¹O₂) production was determined using the Singlet Oxygen Sensor Green reagent (SOSG, Thermo Fisher Scientific, UK) according to the protocol provided by the manufacturer. More details about the assay are described in the supporting information.

Human and murine GBM cells were harvested and incubated in a medium with Z_EGFR:03115-IR700 (30 nM) for 1 h at 4 °C, and samples were analysed using flow cytometry (BD™ LSRII). To test the targeting specificity and internalisation of the conjugate, cells were plated on confocal glass-bottomed dishes (Thermo Fisher Scientific, USA) in complete medium with Z_EGFR:03115-IR700 (1 μM) for 1 h at 4 °C or 1, 3, and 6 h at 37 °C and analysed using a Zeiss LSM700 confocal microscope (Carl Zeiss Inc., Germany). A detailed description of the procedures is given in the supporting information.

U87-MGvIII cells were seeded on petri dishes 24 h before experiments. Afterwards, cells were incubated with Z_EGFR:03115-IR700 (0.1 to 1 μM) for 1, 3, or 6 h at 37 °C. The media were then changed for phenol red-free DMEM medium and cells irradiated (8 or 16 J/cm²) using a LED light source (L690−66−60, Marubeni America Co., USA). Cell viability was determined using the CellTiter-Glo® (Promega, USA) luminescent assay 24 h post-light exposure. To assess ROS production, 5 μM 2′,7′-dichlorofluorescein diacetate (DCFDA; Sigma, UK) was added to phenol-red free medium during irradiation. The cell death at 1, 4, and 24 h post-irradiation was assessed using the Annexin V/Dead Cell Apoptosis Kit (Thermo Fisher Scientific, UK) according to the manufacturer’s instruction. To determine the post-PIT ATP and HMGB1 release, the ENLITEN® ATP assay (Promega, USA) and an HMGB1 ELISA kit (Tecan, IBL International, Germany) were used. Calreticulin exposure on the membrane was measured by flow cytometry (BD™ LSRII). All the methods are described in detail in the supporting information.

The experimental details about co-culturing the immature dendritic cells (iDCs) with PIT-treated U87-MGvIII or DKMG cells are given in the supporting information.

Western blotting was performed as previously described [22]. Proteins released into the medium were extracted using an acetone precipitation protocol (Thermo Fisher Scientific, USA). The list of antibodies used and densitometric analysis are provided in the supporting information.

The preparation of NOTA-Z_EGFR:03115 and its radiolabelling with the ¹⁸F-Al complex was performed as previously described [25].

All experiments were performed in compliance with licences issued under the UK Animals (Scientific Procedures) Act of 1986 and following local ethical review. Studies were compliant with the UK National Cancer Research Institute Guidelines for Animal Welfare in Cancer Research [26] and the ARRIVE (animal research: reporting in vivo experiments) guidelines [27].

The detailed methods are described in the supporting information. Briefly, NCr athymic female mice (5–6 weeks) were bred in-house. C57BL/6J female mice (6–7 weeks) used for the syngeneic model, were purchased from Charles River, UK. The orthotopic GBM U87-MGvIII or BL6-NPE-GFP-Luc mouse models were established as previously described [22, 24]. For the subcutaneous GBM xenografts, U87-MGvIII cells were injected over the right shoulder. Once tumours reached approximately 60 mm³, mice were randomly distributed into the experimental groups.

For PIT treatment studies, subcutaneous and intracranial GBM U87-MGvIII xenografts were randomised into the following treatment groups: (i) light exposure only (100 J/cm²) and (ii) 18 μg Z_EGFR:03115-IR700 with light exposure (100 J/cm², 0.0886 W/cm²). For immunocompetent mice bearing intracranial tumours, 50 J/cm² light dose was used. The tumours were irradiated with a LED light source (L690−66−60, peak 690 ± 20 nm) 1 h post-conjugate i.v. injection. More details are provided in the supporting information.

To monitor the orthotopic tumour growth, mice were imaged using the 1 T M3™ MRI system (Aspect Imaging, Israel) with a T₂-weighted imaging sequence and a dedicated head coil. To perform high-resolution acquisitions, mice were scanned using the 7 T Biospec® horizontal micro-imaging system (Biospec®, Bruker, Germany). The imaging protocols are described in the supporting information.

Mice (n = 5) with MRI-confirmed brain tumours received an i.v. injection of ¹⁸F-AlF-NOTA-Z_EGFR:03115 (12 μg; 2.4 ± 0.15 MBq/mouse), and PET/CT scans were acquired 1, 3, and 5 h post-injection of the radiotracer using an Albira PET/SPECT/CT imaging system. The detailed imaging and data analysis protocols are given in the supporting information.

Dissected tumour and brain tissue samples were collected and immediately embedded in an optimal cutting temperature compound (Tissue-Tek® O.C.T, Netherlands) and snap-frozen in liquid nitrogen.

Further experimental details are given in the supporting information.

In vivo and ex vivo fluorescence images were acquired as stated in the supporting information using an IVIS Spectrum/CT system (Perkin Elmer, USA).

Formalin-fixed brain and tumour tissues were embedded in paraffin, sectioned (5-μm-thick slices), and mounted on microscope slides. Frozen embedded tissues were sectioned into 10-μm-thick slices and mounted on microscope slides before being fixed in ice-cold acetone. The detailed staining procedures with the various antibodies are described in the supporting information.

Tumour and surrounding brain tissue were harvested and dissociated via enzymatic digestion (Liberase TL, Roche, Switzerland). Single-cell suspension was prepared by straining the digested tissue through a 70-μm mesh. Further experimental details are given in the supporting information.

The serum was separated from the whole blood collected from the mice at the 24 h endpoint, snap-frozen, and stored at − 80 °C until further analysis. Concentrations of various cytokines were analysed using a Mouse Cytokine Proinflammatory Focused 10-plex Array (Eve Technologies, Canada).

Unless otherwise stated, data were expressed as the mean ± SD. Statistical significance, sample size calculations, and correlation analysis are described in detail in the supporting information.

The affibody molecule (Z_EGFR:03115), which recognises the murine and human extracellular epitope of EGFR, was conjugated to IR700. Specific and receptor expression-dependent Z_EGFR:03115-IR700 binding (Fig. S1A), as measured by flow cytometry, was in line with the total EGFR level assessed via Western blot (Fig. S1B) in a panel of human and mouse GBM cell lines. In order to confirm that Z_EGFR:03115-IR700 PIT induces target-specific cell death, U87-MGvIII (EGFR high), DKMG (EGFR high), WSz57 (EGFR medium), and U87-MG (EGFR low) cells were incubated with increasing concentrations of the conjugate (0–0.5 μM; 1 h) and exposed to dose of NIR light selected based on our previous studies [22]. A significant decrease in cell viability in a conjugate concentration-dependent manner was seen in both U87-MGvIII and DKMG cells 24 h post-irradiation with 16 J/cm² (Fig. 1A). However, DKMG appeared to be more resistant to the treatment in the presence of low concentrations of the conjugate (survival 75% and 59% at 0.1 and 0.25 μM, respectively) compared to U87-MGvIII cells (survival 36% and 28%, respectively). At the highest concentration of Z_EGFR:03115-IR700 (0.5 μM), both cell lines demonstrated a dramatic loss in cell viability (less than 10% survival). A small but significant reduction in cell viability (81% survival) was observed in U87-MG cells when the highest concentration of the conjugate was tested (0.5 μM). The patient-derived WSz57 cell line was less sensitive to the treatment, most likely due to highly heterogeneous EGFR expression (Fig. S1C). A longer incubation (6 h) of U87-MG, DKMG, and WSz57 cells with the Z_EGFR:03115-IR700 (Fig. S1D) led to an enhanced PIT-mediated cell death that was in line with an increased internalisation of the conjugate as confirmed via confocal microscopy (Fig. S1E).

Further, to determine the mechanism of cell death following the treatment, we used the Annexin V/PI assay. Flow cytometric analysis showed that U87-MGvIII cells were dying rapidly, and within 1 h post-irradiation (0.25 μM conjugate, 8 or 16 J/cm²), there were three distinct cell populations: viable (Annexin⁻/PI⁻; 45-56%), apoptotic (Annexin⁺/PI⁻; 8–12%), and necrotic (Annexin⁺/PI⁺; 35–43%). Importantly, by 24 h post-irradiation, the population of viable cells was 17.5% and 4% (Z_EGFR:03115-IR700 + 8 J/cm² or 16 J/cm² delivered light, respectively) compared to the control groups (91%) (Fig. 1B).

It is well recognised that an essential component of the intracellular pathways that enables ICD and the release of DAMPs is ROS production [21]. Therefore, we subsequently investigated whether Z_EGFR:03115-IR700 PIT-mediated generation of ROS will trigger the activation and trafficking of DAMPs to the extracellular space in vitro. The capability of Z_EGFR:03115-IR700 to induce singlet oxygen (¹O₂) after NIR light activation was initially measured in cell-free conditions. The studies confirmed a significant light dose-dependent SOSG fluorescence enhancement post-irradiation (Fig. S1F). Moreover, U87-MGvIII cells subjected to Z_EGFR:03115-IR700-based PIT (0.25 μM; 16 J/cm²) showed a prominent increase of intracellular ROS production that was significantly suppressed by the ROS scavenger NAC (Fig. 1C). This quenching effect consequently resulted in an inhibition of PIT-induced cell death (Fig. 1D). After PIT with just 8 J/cm², only a slight enhancement in ROS generation was measured in U87-MGvIII cells (Fig. 1C). However, NAC successfully inhibited PIT-induced cell death under each conjugate concentration (Fig. 1D).

Next, we measured the efficacy of Z_EGFR:03115-IR700-based PIT (0.25 μM; 16 J/cm²) to lead to DAMPs (CRT, HMGB1, HSP70, and HSP90) release in U87-MGvIII cells. As shown in Fig. S2A, there was a transient but significant increase in CRT expression level as early as 5 min post-NIR irradiation. We also observed a rapid (5 min) secretion of ATP into the cell culture media post-irradiation (Fig. 2A). Western blot densitometric analysis (Fig. S2) revealed a rapid release of HMGB1, HSP90, and HSP70 into the culture medium after PIT. These strong immunogenic signals were in line with the pronounced U87-MGvIII PIT-induced cell death (Fig. 1A, 0.25 μM; 16 J/cm²) and most likely high sensitivity of these cells to oxidative stress. No other DAMP upregulation was detected in any of the control groups (Fig. 2B; Fig. S2). Additionally, ELISA results (corroborated by Western blot data) showed that cells irradiated 1 h after Z_EGFR:03115-IR700 treatment released HMGB1 in a time-dependent manner when compared to the control cells (Fig. 2C). We then investigated whether the enhanced levels of ICD markers, induced by Z_EGFR:03115-IR700 PIT in U87-MGvIII cells, would trigger phenotypic maturation of DCs in a similar manner to conjugates reported by others [28]. We co-cultured Z_EGFR:03115-IR700-PIT-treated U87-MGvIII cells with iDCs for 48 h. Subsequent flow cytometry analysis showed a significant increase in the expression of CD86 (Fig. 2D) and MHC class II (HLA-DR) (Fig. 2E) molecules on the surface of DCs exposed to PIT-treated U87-MGvIII cells compared to controls. However, there was no change in the CD40 expression level (Fig. 2F).

The capability of Z_EGFR:03115 to target EGFR-expressing cells in vivo was evaluated using an orthotopic U87-MGvIII tumour model (Fig. 3). Five days post-cell implantation, the progression of intracranial malignancies was assessed by T₂-weighed MRI (Fig. 3A). To confirm the targeting abilities of Z_EGFR:03115, PET/CT studies were performed. Images were acquired at 1, 3, and 5 h post-administration using the well-established EGFR-targeting imaging agent ¹⁸F-AlF-NOTA-Z_EGFR:03115 (Fig. S3A) (n = 3) [25]. The images recorded 3 h post-injection demonstrated preferential and focal accumulation of the radioconjugate in the tumour mass (Fig. 3A and Fig. S3A-B). Negligible activity was observed in the normal cerebral tissue that provided sharp delineation of the tumours with high tumour/parenchyma contrast. The ROI quantitative analysis showed a time-dependent increase of tumour radioactivity uptake with the highest value observed 5 h p.i. (%ID/g₅₀ = 5.14 ± 1.17) (Fig. 3B). H&E staining of axial brain sections confirmed the presence of well-defined tumour masses, which were in line with PET/CT and MRI signals in vivo as well as radioactivity signals measured ex vivo (Fig. S3B). When Z_EGFR:03115-IR700 was administered i.v., the strong fluorescence signal of the conjugate was detected ex vivo within the brain EGFR-positive lesions (2–3 mm in diameter) as early as 1 h post-injection in mice bearing MRI-confirmed tumours (Fig. 3C) (n = 3).

To assess whether Z_EGFR:03115-IR700-PIT shows an anti-tumour effect against GBM in vivo, subcutaneous and intracranial U87-MGvIII tumours were established in nude mice. In contrast to our previous studies using three Z_EGFR:03115-IR700-PIT doses over three consecutive days [22], herein, we tested whether administration of just one dose would inhibit tumour growth. The conjugate (18 μg) was injected intravenously (Fig. S4A), and the subcutaneous tumours were irradiated 1 h later with 100 J/cm². A significant delay in tumour growth was observed during the initial 7 days in the group receiving Z_EGFR:03115-IR700-PIT compared to the control (light only 100 J/cm²) (Fig. S4B). However, due to the re-growth of measurable tumours at this point, a second PIT dose was delivered. Unfortunately, no further growth inhibition was observed (Fig. S4B). Next, we studied the early anti-tumour response to PIT using the orthotopic U87-MGvIII model by MRI. Following tumour establishment, T₂- and T₂*-weighted images were acquired at baseline and 1 or 4 h post-Z_EGFR:03115-IR700-PIT (18 μg; 100 J/cm²). T₂*-weighted imaging was selected because of its sensitivity to the presence of paramagnetic species such as deoxyhaemoglobin and its capability to detect cerebral and intratumoural micro-haemorrhage under pathologic conditions. T₂*-weighted images showed an intratumoural signal decrease corresponding to haemorrhage and hemosiderin deposition 1 h post-NIR irradiation in mice treated with Z_EGFR:03115-IR700-PIT as compared to the control group (Fig. 4A). We also observed an enlargement of the lesions on T₂-weighted images caused by direct cytotoxic effects on tumour cells, damage to the tumour vasculature, and induction of an inflammatory reaction post-PIT, which resulted in the swelling of the surrounding brain tissue and a mass effect (Fig. 4A). Furthermore, increased signal intensity on T₂-weighted images of parenchyma surrounding the tumour growing from the top of the skull along the pathway of cell implantation most likely corresponds to the cerebral oedema formation following PIT (Fig. 4A, yellow and blue arrows). The difference between areas of increased T₂-weighted signal intensity in the surrounding tumour brain parenchyma (Fig. 4A, yellow vs blue arrows) was probably due to the changes in light distribution. The intratumoural quantification of R₂* (Fig. 4B, C) indicated that mice exposed to Z_EGFR:03115-IR700-PIT had a higher percentage of changes in R₂* values compared to the controls (3.1% (n = 3) for the control group vs 11.3% (n = 3) and 41.1% (n = 2) for 1 h and 4 h groups post-PIT, respectively), which effectively confirmed an acute tumour response to PIT. Hypointense cores highlighted after PIT are compliant with early necrosis. These changes were consistent with H&E staining of the brain sections showing tumour necrosis and micro-haemorrhage patterns associated with pyknosis and apoptotic bodies on the margins of the PIT-treated tumours (Fig. 5, Fig. S4C). The tumour necrosis became extensive 24 h post-PIT, showing the strong effect of PIT especially on the core of U87-MGvIII tumours. Moreover, visual assessment of IHC staining for Ki67 indicated reduced cell proliferation 24 h post-conjugate irradiation. Our data also indicate that Z_EGFR:03115-IR700-PIT triggered a substantial release of HSP70 within 1 h after treatment (Fig. 5).

We used the BL6-NPE-GFP-Luc syngeneic model to investigate whether a T cell-focused immune response can be elicited by light-activated Z_EGFR:03115-IR700 in the brain setting (18 μg/50 J/cm²; Fig. 6A). In this case, we decided to lower the light intensity as we observed in some mice bearing intracranial U87-MGvIII tumours that the mass effect led to a significant deterioration of their condition. Flow cytometry analysis of tumour samples at 24 h post-PIT (18 μg/50 J/cm²) showed higher levels of CD4+ and CD8+ immune cells in tumours exposed to PIT compared to the control groups (Fig. 6B). The detailed gating strategy is implemented in Fig. S5A. A pronounced number of CD8+ cells was also detected on IHC slices of treated tumour (Fig. S5B). A similar but less pronounced trend was observed in CD69 expression level on T cells, an early activation antigen involved in the transmission of costimulatory signals (Fig. 6B). Thereafter, we measured the level of selected pro-inflammatory cytokines which have the ability to enhance the immune response towards tumours by activating NK cells, CD8+ T cells, and macrophages. We found a significant increase of IL-6 and an upward trend of IL-1β, TNF-α, and IL-12 levels from the control groups to the Z_EGFR:03115-IR700-PIT group, while the level of IL-10, indicating immunosuppression revealed no change regardless of the treatment group (Fig. 6C).

Finally, to check the effect of PIT on the PD-1/PD-L1 axis, we assessed whether Z_EGFR:03115-IR700-PIT induces changes in the PD-L1 expression on tumour cells by flow cytometry. We initially confirmed that IFN-γ stimulation leads to a considerable increase in PD-L1 expression in multiple GBM cell lines (Fig. S5C). Subsequently, we detected a significant decrease of PD-L1 in U87-MGvIII cells in response to Z_EGFR:03115-IR700-PIT with and without IFN-γ stimulation compared to the controls in vitro (Fig. S5D-E). These results were next corroborated by in vivo findings showing a downregulation of PD-L1 also on the surface of BL6-NPE-GFP-Luc cells post-PIT (Fig. 6D).