RPS 906 - Exploring the frontiers in hybrid and molecular imaging

Author Block: W. Zhang, X. Liang, Y. Du, J. Tian, N. Hong; Beijing/CN
Purpose: Albumin-bound paclitaxel and gemcitabine (AG) chemotherapy is a mainstay in treatment of pancreatic ductal adenocarcinoma (PDAC), unfortunately not all patients respond to this treatment. Clinical imaging techniques cannot precisely evaluate and predict the response to AG therapies over several weeks. A strong fibrotic reaction is a hallmark of drug-resistance while depletion of fibrosis is a positive response to AG. Extradomain-B fibronectin (EDB-FN) is an important element of fibrosis in PDAC. Here, we prepared EDB-FN targeted Gd-based contrast agent (EDB-Gd) to perform molecular MRI for early, noninvasive and quantitative assessment of treatment response in PDAC.
Methods or Background: BxPC-DR was pre-treated with AG to establish acquired drug resistance. Subcutaneous and orthotopic models with BxPC-DR or BxPC were established. Mice were intravenously injected with EDB-Gd or Gd-DOTA. The ratio of T1 value reduction (T1d%) were compared quantitatively. For chemotherapy monitoring, MRI was performed before and after AG treatments. Histological analyses were used for validation.
Results or Findings: Molecular MRI with EDB-FN could specifically detect and quantify fibrogenesis in PDAC xenografts at a low dose 0.05mmol/kg, which is half of clinical dosage of Gd. The optimal imaging time point was 30min after injection of EDB-Gd. In addition, the targeted probe generated more robust contrast-enhanced and longer retention compared to traditional Gd-DOTA. For chemotherapy montoring, T1d% were significantly increased 2.5-fold in drug-resistance xenografts group in fibrotic tumor areas compared to AG-sensitive group (p < 0.05). Comparing the T1d% before and 5 days after AG predicted treatment response.
Conclusion: This study indicates EDB-FN-targeted molecular MRI possesses clinical applications in accurate assessment and prediction of AG chemotherapy.
Limitations: Correlations between the observed fibrotic changes on MRI with pathologic markers of treatment response and even survival and prognosis are needed in the future.
Funding for this study: This study was funded by the Beijing Natural Science Foundation (Grant No. 7244524, 7212207) and National Natural Science Foundation of China (Grant Nos. 62027901, 82272111, 92159303, 82071896, 81871422, 81871514, and 81227901).
Has your study been approved by an ethics committee? Yes
Ethics committee - additional information: All experimental studies were approved by the Ethics Committee of the Peking University People's Hospital (2024PHE048).

Author Block: A. Helfen, M. Mallik, M. Stölting, E. Hoffmann, C. Höltke; Münster/DE
Purpose: A significant prognostic factor of tumor malignancy is the formation of a hypoxic supportive tumor microenvironment (TME). The endothelin (ET)M signaling network is linked to tumor hypoxia through stabilized hypoxia-inducible factor 1 in a feedback loop. Here, we examined the interrelation of both cellular signaling systems by multispectral optoacoustic tomography (MSOT).
Methods or Background: Murine syngeneic 4T1 breast tumors were examined in vivo using MSOT depicting deoxygenated (Hb) and oxygenated (HbO2) hemoglobin content to detect hypoxic regions over one week. An exogenous fluorescent endothelin-A receptor (ETAR) probe served for evaluating ETAR expression status. Therapeutic interventions (anti-angiogenic and macrophage depletion) were evaluated concerning Hb/HbO2 ratio and ETAR expression changes. Treatment response to Bevacizumab, Clodronate and Sorafenib was detected over one week.
Results or Findings: MSOT was capable of delineating and quantifying hypoxia within tumor lesions. 4T1 tumors were highly hypoxic compared to healthy tissue, represented by oxygen saturation (sO2) of 0.33 vs. 0.79, respectively. Baseline data of the ETAR probe showed an initial rise in signal intensity from day 0 to day 8, corresponding to tumor growth. Therapeutic interventions showed that the ETAR signal intensity could be significantly reversed, while all applied therapies did not lead to significant tumor growth reduction. However, Sorafenib and Bevacizumab led to a significant increase in sO2 values. MSOT data were supported by subsequent immunohistochemistry.
Conclusion: Tumor hypoxia within syngeneic murine breast cancer can be visualized non-invasively by MSOT evaluating hemoglobin (oxygenated and deoxygenated ratios) as well as ETAR expression. Furthermore, MSOT was able to depict therapeutic effects already in the early course of treatment representing a potential imaging biomarker.
Limitations: Limited penetration depth of MSOT is significantly improved compared to optical imaging, but with regard to translation still mainly suitable for superficial tissues.
Funding for this study: Financial support from the German Research Foundation (DFG, SF656 A04), from the medical faculty of the University of Münster (IMF: I-HÖ111709) and from the Joachim Herz Stiftung is gratefully acknowledged.
Has your study been approved by an ethics committee? Not applicable
Ethics committee - additional information: All animal experiments described in this study were approved by the responsible authorities (“Landesamt für Natur, Umwelt und Verbraucherschutz NRW”, Germany, Protocol No. 84-02.04.2017.A011).

Author Block: P. Akl, A. Carret, A. Houmeau, A. Gautheron, I. Goddard, J-B. Langlois, B. Montcel, F. Lerouge, P. C. Douek; Lyon/FR
Purpose: Photodynamic therapy (PDT) uses light to activate photosensitizers (PS) but is limited by shallow penetration. X-ray PDT (XPDT) overcomes this by using X-rays for deeper activation. Spectral Photon-Counting Computed Tomography (SPCCT) enhances imaging with K-Edge capabilities. This study aims to optimize XPDT in vitro and in vivo using SPCCT and gadolinium nanoparticles (GdNp) as contrast and therapeutic agents.
Methods or Background: GdNp doped with terbium were synthesized, functionalized for biocompatibily with different coatings (PEG and Silica) and the photosensitizer, rose bengal (RB) and irradiated at different concentrations (0.02-1 M) in vitro using SPCCT with different X-ray dose parameters (80-140 KVp and 10-300 mAs). Luminescence emission was recorded using optical fiber immersed in the GdNp solution. 63 athymic nude mice with MDA-MB-231 cell-derived breast cancer xenograft models were irradiated using SPCCT(120kVp and 300mAs) 60 axial acquisitions/ 1 sec, meandose 30mGy, 1800mGy in total ,24 hours after intra tumoral injection. Safety, biodistribution, and therapeutic effects were assessed through mice examination every 3 days for tumor volume to assess tumor growth delay .Biodistribution was monitored using SPCCT imaging.
Results or Findings: Fluorescence emission increased with X-ray dose and concentration of GdNp. SPCCT imaging revealed specific distribution of GdNp , with luminescence emission proportional to the administered dose in vitro and in vivo. The silica + RB nanoparticle group showed a slight improvement in efficacy compared to controls, with delayed tumor growth. Biodistribution studies indicated relatively different patterns of intratumoral and peritumoral localization, and heterogenous distribution.
Conclusion: This technology shows potential for customizing and enhancing the efficacy of XPDT , modulate luminescence intensity by adjusting X-ray dose parameters and nanoparticle concentration. Silica + RB showed a slight improvement in efficacy compared to controls.
Limitations: Though further studies are required to confirm significance.
Funding for this study: EU Horizon 2020 grant agreement 899549
Has your study been approved by an ethics committee? Yes
Ethics committee - additional information: ethical comittee IRB APAFIS#44558.

Author Block: M. J. Antons, S. Kloiber-Langhorst, H. Hirner-Eppeneder, F. Herr, S. Ziegler, M. Brendel, J. Ricke, M. Heimer, C. C. Cyran; Munich/DE
Purpose: Three-time point [18F]FDG-PET/CT imaging allows for in vivo monitoring of a combined anti-PD-L1/anti-CTLA-4 immunotherapy in a murine melanoma model validated by time point-matched multiparametric immunohistochemical reference standard
Methods or Background: Melanoma cells (B16-F10) were injected subcutaneously into the abdominal flank of C57BL/6 mice (n=40). Following a baseline scan after day 7, the therapy group received 5 injections (i.p.) of anti-PD-L1 and anti-CTLA-4 antibodies on days 7, 9, 11, 13 and 15. The control group received sham treatment. Follow-up scans were performed on day 13 and 19. Tumor allografts were harvested for time point-matched immunohistochemistry (CD8, Ki-67, TUNEL) to validate PET/CT parameters (MTV, SUVmax) as imaging biomarkers of early therapy response.
Results or Findings: At follow-up 1 (FU-1), the therapy group exhibited significantly lower MTV compared to the control group (p=0.0037). By follow-up 2 (FU-2), both MTV and SUVmax were significantly lower in the therapy group versus the control group (MTV: p=0.0078; SUVmax: p=0.00034). Ex vivo analysis revealed significant anti-tumor effects in the therapy group, with a significantly higher apoptosis rate at FU-1 (p= 0.012) and FU-2 (p= 0.001). Moreover, the therapy group demonstrated a significant increase in CD8-positive T-cells at FU-2 (p=0.0027), while tumor cell proliferation was significantly lower at both follow-up time points (FU-1: p=0.012; FU-2: p=0.012).
Conclusion: Multi-time point [18F]FDG-PET/CT allowed for the early non-invasive monitoring of a combined immunotherapy with anti-PD-L1/anti-CTLA-4 in experimental melanomas, validated by multiparametric immunohistochemistry. The significantly lower tumor glucose metabolism was paralleled by significant pro-immunogenic, pro-apoptotic and anti-proliferative effects of the combined immunotherapy.
Limitations: The allograft model of melanoma may have limited translational relevance to human tumor pathophysiology. Secondly, the observation period was relatively short, and no clinical endpoints such as overall survival of the animals were determined.
Funding for this study: None
Has your study been approved by an ethics committee? Yes
Ethics committee - additional information: All animal experiments were performed in accordance with the guidelines for the use of living animals in scientific studies and the animal study was officially approved (ROB-55.2-2532.Vet_02-19-32).

Author Block: S. J. Bartsch¹, J. Friske¹, M. Hacker¹, D. Laimer-Gruber¹, D. Prinz¹, T. Wanek¹, T. H. Helbich¹, K. Pinker-Domenig²; ¹Vienna/AT, ²New York, NY/US
Purpose: Hypoxia is a driver of breast cancer (BC) progression, inducing more aggressive phenotypes and intratumoral neovascularization. The quantification of hypoxia and neovascularization with simultaneous multiparametric [18F]FMISO-PET/MRI would benefit the characterization of the hypoxic tumor microenvironment in BCs.
We aim to combine simultaneous multiparametric [18F]FMISO-PET/MRI biomarkers and use them for the identification of intratumoral clusters for a holistic assessment of hypoxia and neovascularization in BCs.
Methods or Background: Female athymic nude mice (n = 32) were inoculated with luminal A, HER2+ or triple negative BC cells. PET/MRI was performed on a Bruker 94/30USR system, combined with a Bruker PET-insert.
Hypoxia was evaluated using [18F]FMISO-PET, and by hyperoxic blood oxygen level dependent (BOLD) MRI. Neovascularization was assessed via dynamic contrast enhanced MRI, and non-contrast-enhanced intravoxel incoherent motion MRI. Intratumoral clusters were identified based on a hierarchical cluster analysis using R (version 4.2.3).
Results or Findings: The cluster analysis of [18F]FMISO-PET/MRI revealed distinct clusters in all BC subtypes. Clusters corresponding to hypoxia showed elevated SUVbw values and the lowest ktrans and ve, indicating dense tissue and limited vessel permeability, along with little change in R2* following hyperoxic BOLD-MRI. The least hypoxic cluster had the lowest SUVbw values and the highest ktrans and ve of all clusters and high change in R2*. Hypoxic clusters were more prevalent in HER2+ and triple negative BCs than in luminal A BCs, while less hypoxic clusters were most common in luminal A BCs.
Conclusion: Simultaneous multiparametric [18F]FMISO-PET/MRI provides a holistic perspective on hypoxia and neovascularization in three BC molecular subtypes. These insights enable a non-invasive characterization of BC, and may be used for assessing treatment response.
Limitations: The use of intratumoral cluster identification for the assessment of treatment response has to be confirmed in upcoming studies.
Funding for this study: This work was funded by the Vienna Science and Technology Fund (WWTF), project number LS19-018.
Has your study been approved by an ethics committee? Yes
Ethics committee - additional information: This animal study was approved by Austrian Federal Ministry of Education, Science and Research [66.009/0284-WF/V/3b/2017; 2020-0.363.124; 2022-0.726.820] and the Intramural Committee for Animal Experimentation of the Medical University of Vienna.

Author Block: L. Brizzi, L. Preda, C. Bortolotto, S. Megalizzi, D. Malerba, F. Checchin; Pavia/IT
Purpose: The study aimed to compare radiomic features between conventional in vivo CT and micro-CT of ex vivo lung tumor blocks following lobectomy. This analysis included 60 patients with lung cancer, comprising 30 adenocarcinomas (ADK) and 30 squamous cell carcinomas (SCC). The goal was to assess the variation and significance of features to improve predictive accuracy in distinguishing between tumor types.
Methods or Background: The dataset comprised 107 radiomic features, compliant with IBSI standards, extracted using Pyradiomics software. Statistical analyses, including a percentage variation calculation and t-tests, were performed to evaluate the correlation and consistency of features between in vivo CT and ex vivo micro-CT. The analysis focused on features with potential utility in discriminating between ADK and SCC tumors.
Results or Findings: Out of the 107 radiomic features, 82 showed less than 10% variation between the two imaging modalities, with 46 features exhibiting a variation of less than 1%. Shape features and approximately 90% of GLCM features demonstrated strong consistency. T-tests revealed that 21 radiomic features had a p-value < 0.05, indicating statistical significance. These features included various shape characteristics and first-order statistics that are crucial for tumor classification.
Conclusion: The findings indicate a strong correlation between the radiomic features extracted from in vivo CT and micro-CT, particularly in shape and GLCM features. The identified significant features offer promising potential for improving predictive models for lung cancer classification between ADK and SCC.
Limitations: The study was limited to a specific subset of lung tumors (ADK and SCC), and the generalizability to other lung cancer subtypes remains uncertain. Additionally, technical variations in CT acquisition parameters might affect feature extraction consistency. Further validation with larger datasets is necessary.
Funding for this study: Founding are provided by Research Foundings on AI of the IRCCS Policlinico San Matteo
Has your study been approved by an ethics committee? Yes
Ethics committee - additional information: Protocol 25657/2024