Terbium-161 (Tb-161) emits internal conversion and Auger electrons, in addition to beta-minus radiation, which might be of added benefit for targeted radionuclide therapy (TRT) compared to Lutetium-177 (Lu-177). We extensively compared Lu-177 and Tb-161 for fibroblast activation protein (FAP)-targeted TRT in a preclinical setting. To study this, FAP-2286 was labeled with Lu-177 and Tb-161 and characterized in vitro on FAP-expressing cells and ex vivo using patient tumor samples. Moreover, in vivo studies (i.e. biodistribution and efficacy) were performed using a clinically representative pancreatic ductal adenocarcinoma (PDAC) mouse model. Biodistribution was performed 1, 4, 24, and 48 h post injection of 5 MBq/500 pmol [177Lu]Lu-FAP-2286 or [161Tb]Tb-FAP-2286. Subsequently, animals were treated with 4 × 40 MBq/500 pmol [177Lu]Lu-FAP-2286 or [161Tb]Tb-FAP-2286 and with alternating doses of 2 × 40 MBq/500 pmol of each radiopharmaceutical. No difference in [177Lu]Lu-FAP-2286 and [161Tb]Tb-FAP-2286 uptake was observed in the cell models. In vivo studies did not show a survival benefit after 4 × 40 MBq/500 pmol [177Lu]Lu-FAP-2286 or [161Tb]Tb-FAP-2286, while Kaplan-Meier analyses demonstrated a modest prolonged survival after tandem therapy in mice that first received [177Lu]Lu-FAP-2286 followed by [161Tb]Tb-FAP-2286. Dosimetry calculations based on autoradiography studies on patient tumor samples showed that even with lower binding, a higher absorbed dose to the tumor can be accomplished with [161Tb]Tb-FAP-2286. In our in vitro and in vivo studies, [177Lu]Lu-FAP-2286 and [161Tb]Tb-FAP-2286 demonstrated similar behavior. In the applied PDAC mouse model, FAP-TRT showed limited therapeutic efficacy, most likely due to the limited radiopharmaceutical uptake observed in the tumors. This hampered determination of a potential benefit of either radioisotope for FAP-TRT. Of note, a modest response was observed in the tandem therapy group that first received [177Lu]Lu-FAP-2286, followed by [161Tb]Tb-FAP-2286.

Gastrin releasing peptide receptor (GRPR) gained interest for targeted radionuclide therapy (TRT) of prostate cancer (PCa). However, its expression in advanced disease remains understudied. We correlated clinical, biological, molecular and pathological characteristics, with GRPR expression in PCa patient-derived xenograft (PDX) models, and compared it with prostate specific membrane antigen (PSMA) expression. GRPR expression was studied on tissue microarrays of 107 PDXs using immunohistochemistry. Seventy-six samples were GRPR positive. There were no differences in GRPR expression between primary and metastatic lesions, and androgen-naïve, androgen sensitive and castration resistant samples. High GRPR expression was frequent in androgen receptor (AR) positive tumors, high PSMA-expressing tumors, tumors without phosphatase and tensin homolog (PTEN) loss, and vimentin negative tumors. AR-positivity was the most important predictor of high GRPR expression. In PSMA negative tumors, GRPR expression was high in chromogranin positive tumors, tumors without PTEN loss, in absence of retinoblastoma1-gene mutation, and high vimentin-expressing tumors. Among the neuroendocrine PCa’s (NEPC), 14/20 were GRPR and 3/20 were PSMA positive. All PSMA positive tumors were GRPR positive. Among metastatic NEPC samples, 9/10 and 1/10 were GRPR and PSMA positive, respectively. Based on the aforementioned, we conclude that GRPR is a relevant target for advanced PCa. Moreover, the selective GRPR expression in NEPC opens new avenues for GRPR-TRT.

BACKGROUND: Preclinically, radiopharmaceuticals are currently mainly evaluated in two-dimensional (2D) cell models, which lack clinical features that are relevant for accurate evaluation of targeted radionuclide therapy (TRT) responses. The development of three-dimensional (3D) cell models in the last years offers opportunities to overcome at least parts of the limitations of 2D cell models, but such 3D cell models are currently only rarely used in nuclear medicine research. Moreover, the comparison between 2D and 3D cell models, aimed at demonstrating the potential added value of 3D cell models, is even more scarce. To fill this gap and promote the use of more clinically relevant 3D cell models in nuclear medicine research, we performed such a comparative study. For this, we developed and evaluated 3D cell models derived from the prostate-specific membrane antigen (PSMA)-expressing human cancer cell lines LNCaP and PC3-PIP, by culturing these cells in anti-adhesive round bottom plates (“bio-spheroids”) or by culturing LNCaP cells in Matrigel (MG) or Noviogel-P5K (NG) domes (”MG-spheroids” or “NG-spheroids”). Hereafter, PSMA expression levels and [111In]In-PSMA-I&T uptake were determined and compared between 3D and 2D cell models. Additionally, we assessed cell viability of 3D- versus 2D-cultured cells after external beam radiation therapy (EBRT) and PSMA-TRT using [177Lu]Lu-PSMA-I&T. RESULTS: No significant differences in viability were observed between bio-spheroids versus 2D cell models after EBRT and PSMA-TRT, neither in LNCaP nor in PC3-PIP cells. In contrast, LNCaP MG-spheroids had a significantly better response to PSMA-TRT in comparison to the 2D-cultured cells. This was despite a lower PSMA expression level and lower [111In]In-PSMA-I&T uptake in the MG-spheroids. Importantly, no significant difference in radiosensitivity was observed between these MG-spheroids and the 2D cell model. CONCLUSIONS: Despite lower PSMA expression levels and lower radiopharmaceutical uptake in LNCaP MG-spheroids, and albeit similar radiosensitivity, PSMA-TRT induced a stronger reduction in viability in the MG-spheroids in comparison to the 2D-cultured cells. In contrast, no differences were observed in PSMA-TRT efficacy between bio-spheroids and the 2D cell model. The aforementioned leads to the hypothesis that biological factors important for TRT, e.g. cross-radiation and radiopharmaceutical retention within the 3D cell structure, are better represented in MG-spheroids where cell-cell interactions are already formed prior to radiopharmaceutical incubation.

BACKGROUND:Targeted radionuclide therapy (TRT) directed at fibroblast activation protein (FAP), highly expressed on cancer-associated fibroblasts (CAFs), is a promising approach for treating stroma-rich tumors such as pancreatic ductal adenocarcinoma (PDAC) and breast cancer (BC). To better assess the efficacy of FAP-TRT and to get more insight into the potential of crossfire effects from target expressing CAFs to neighboring cancer cells, more representative preclinical models are needed. Therefore, we established a direct 2D co-culture model consisting of both CAFs and cancer cells. MATERIALS & METHODS:We developed a clinically representative PDAC and BC 2D co-culture model by co-seeding human CAFs and matching cancer cells. First, the CAFs and cancer cells were transduced with distinct fluorescent and bioluminescent reporter genes (RFP-CBG99 and GFP-CBR2, respectively). Fluorescent microscopy enabled the optimization of the seeding densities. Patient tumor samples were analyzed to guide the co-culture design, ensuring realistic representation of the patient situation. Co-cultures were optimized and subsequently treated with external beam radiation therapy and radionuclide therapy (i.e. lutetium-177). The luciferase expressed by the reporter gene enabled monitoring of CAF and cancer cell viability. Cell survival was assessed using dual-color bioluminescence imaging (BLI) at 540SP, 600LP, and open filter settings. RESULTS:The established PDAC and BC co-culture models closely mimicked patient tumors with regard to CAF localization, stroma density, and FAP expression. The dual-color BLI allowed for reliable discrimination of CAF and cancer cell viability. We observed a drastic decrease in cell viability after EBRT in all four cell lines, whereas only three out of four cell lines responded to lutetium-177 therapy. In addition, all cell lines exhibited similar treatment responses in the co-culture and monoculture settings. CONCLUSIONS:We successfully established 2D co-culture models of PDAC and BC that include CAFs neighboring the cancer cells, as is the case in the patient tumor microenvironment. The use of dual-color reporter genes enabled efficient, cell-type-specific analysis of radionuclide therapy efficacy via BLI. The developed models provide a user-friendly platform for evaluating therapeutic responses and can be further refined into 3D systems for even higher patient resemblance.

Cancer remains a leading cause of mortality globally, driving ongoing research into innovative treatment strategies. Preclinical research forms the base for developing these novel treatments, using both in vitro and in vivo model systems that are, ideally, as clinically representative as possible. Emerging as a promising approach for cancer management, targeted radionuclide theranostics (TRT) uses radiotracers to deliver (cytotoxic) radionuclides specifically to cancer cells. Since the field is relatively new, more advanced preclinical models are not yet regularly applied in TRT research. This narrative review examines the currently applied in vitro, ex vivo and in vivo models for oncological research, discusses if and how these models are now applied for TRT studies, and whether not yet applied models can be of benefit for the field. A selection of different models is discussed, ranging from in vitro two-dimensional (2D) and three-dimensional (3D) cell models, including spheroids, organoids and tissue slice cultures, to in vivo mouse cancer models, such as cellline-derived models, patient-derived xenograft models and humanized models. Each of the models has advantages and limitations for studying human cancer biology, radiopharmaceutical assessment and treatment efficacy. Overall, there is a need to apply more advanced models in TRT research that better address specific TRT phenomena, such as crossfire and abscopal effects, to enhance the clinical relevance and effectiveness of preclinical TRT evaluations.

Peptide-based radiopharmaceuticals targeting neurotensin-receptor-1 (NTS1) are mainly stabilized using chemical modifications at the NT[8-13] sequence, thus increasing the stability and the uptake of the corresponding radionuclide-macrocycle-linker-bioconjugate. We postulate that the introduction of the linker at the N-term part induces additional cleavage sites that can be further stabilized to achieve a prolonged uptake. Double (JMV 7259 and JMV 7222) and triple-stabilized neurotensin analogues (JMV 7258 and JMV 7490) were synthesized, radiolabeled, and evaluated on HT-29 cells (NTS1+). Nanomolar NTS1-affinity and high internalization rates were observed for all of the radiopharmaceuticals. Efflux was lower for radiolabeled JMV 7490. Consequently, [111In]In-JMV 7490 showed uptake of 5.86 ± 0.86 and 3.65 ± 0.29% ID/g of tissue in HT-29 xenografts at 1 and 4 h, respectively. We have successfully shown that high and persistent uptake of NTS1-positive tumor cells is achievable by stabilization of the N-term part. Efflux also appears to be a critical parameter for the successful targeting of NTS1 using radiopharmaceuticals.

Gastrin releasing peptide receptor (GRPR)-targeting radiotracers have been studied (pre)-clinically with promising results. Patients eligible for this treatment are likely to have undergone prior treatments with other anti-cancer agents, including chemotherapy. Chemotherapies are known to alter cancer cell’s gene expression and radiosensitivity, potentially impacting GRPR expression and the response to radionuclide therapy. We studied the effect of two commonly applied chemotherapies, doxorubicin (DXR) and docetaxel (DTX), on GRPR expression, GRPR radiotracer uptake, and response to external beam radiation therapy (EBRT) and targeted radionuclide treatment, in prostate cancer (PCa) and breast cancer (BC) cells. Additionally, in-vivo uptake of the GRPR-targeting radiotracer “NeoB” in PC-3 and T47D xenograft-bearing mice was assessed using SPECT/CT following chemotherapy treatment. DTX significantly decreased GRPR expression, radiotracer uptake, and radiosensitivity of PC-3 cells in-vitro. DXR pre-treated T47D cells demonstrated an increased GRPR expression and radiotracer uptake, and were less sensitive to EBRT. In-vivo, DTX pre-treatment increased [177Lu]Lu-NeoB uptake in PC-3 xenografts, but this was not GRPR mediated. DXR pre-treatment did not alter [177Lu]Lu-NeoB uptake in T47D xenografts, but an increase in GRPR mRNA expression was observed. Our data demonstrated that chemotherapy alters mechanisms relevant for the success of GRPR-mediated radionuclide therapy in PCa and BC cells in-vitro. These finding were less prominent in-vivo and additional studies are needed to unravel this.

Background Terbium-161 (Tb-161) emits internal conversion and Auger electrons, in addition to beta-minus radiation, which might be of added benefit for targeted radionuclide therapy (TRT) compared to Lutetium-177 (Lu-177). We extensively compared Lu-177 and Tb-161 for fibroblast activation protein (FAP)-targeted TRT in a preclinical setting. To study this, FAP-2286 was labeled with Lu-177 and Tb-161 and characterized in vitro on FAP-expressing cells and ex vivo using patient tumor samples. Moreover, in vivo studies (i.e. biodistribution and efficacy) were performed using a clinically representative pancreatic ductal adenocarcinoma (PDAC) mouse model. Biodistribution was performed 1, 4, 24, and 48 h post injection of 5 MBq/500 pmol [ 177 Lu]Lu-FAP-2286 or [ 161 Tb]Tb-FAP-2286. Subsequently, animals were treated with 4×40 MBq/500 pmol [ 177 Lu]Lu-FAP-2286 or [ 161 Tb]Tb-FAP-2286 and with alternating doses of 2×40 MBq/500 pmol of each radiopharmaceutical. Results No difference in [ 177 Lu]Lu-FAP-2286 and [ 161 Tb]Tb-FAP-2286 uptake was observed in the cell models. In vivo studies did not show a survival benefit after 4×40 MBq/500 pmol [ 177 Lu]Lu-FAP-2286 or [ 161 Tb]Tb-FAP-2286, while Kaplan-Meier analyses demonstrated modestly prolonged survival after tandem therapy, in mice that first received [ 177 Lu]Lu-FAP-2286 followed by [ 161 Tb]Tb-FAP-2286. Dosimetry calculations based on autoradiography on patient tumor samples showed that even with lower binding, a higher absorbed dose to the tumor can be accomplished with [ 161 Tb]Tb-FAP-2286. Conclusions In our vitro and in vivo studies, [ 177 Lu]Lu-FAP-2286 and [ 161 Tb]Tb-FAP-2286 demonstrated similar behavior. In the applied PDAC mouse model, FAP-TRT showed limited therapeutic efficacy, with a modest response observed in the tandem therapy group that first received [ 177 Lu]Lu-FAP-2286, followed by [ 161 Tb]Tb-FAP-2286.

BackgroundFibroblast activation protein (FAP), a transmembrane serine protease overexpressed by cancer-associated fibroblasts in the tumor stroma, is an interesting biomarker for targeted radionuclide theranostics. FAP-targeting radiotracers have demonstrated to be superior to [18F]FDG PET/CT in various solid cancers. However, these radiotracers have suboptimal tumor retention for targeted radionuclide therapy (TRT). We aimed to develop a novel FAP-targeting pharmacophore with improved pharmacokinetics by introducing a substitution at the 8-position of (4-quinolinoyl)-glycyl-2-cyanopyrrolidine, which allows for conjugation of a chelator, dye, or other payloads.ResultsHere we showed the synthesis of DOTA-conjugated eFAP-6 and sulfo-Cyanine5-conjugated eFAP-7. After chemical characterization, the uptake and specificity of both tracers were determined on FAP-expressing cells. In vitro, [111In]In-eFAP-6 demonstrated a superior affinity and a more rapid, although slightly lower, peak uptake than gold standard [111In]In-FAPI-46. Confocal microscopy demonstrated a quick FAP-mediated internalization of eFAP-7. Studies with HT1080-huFAP xenografted mice confirmed a more rapid uptake of [177Lu]Lu-eFAP-6 vs. [177Lu]Lu-FAPI-46. However, tumor retention at 24 h post injection of [177Lu]Lu-eFAP-6 was lower than that of [177Lu]Lu-FAPI-46, hereby currently limiting its use for TRT.ConclusionThe superior affinity and faster tumor accumulation of eFAP-6 over FAPI-46 makes it a suitable compound for radionuclide imaging. After further optimization, the eFAP series has great potential for various oncological interventions, including fluorescent-guided surgery and effective targeted radionuclide theranostics.

Multimodality reporter gene imaging combines the sensitivity, resolution and translational potential of two or more signals. The approach has not been widely adopted by the animal imaging community, mainly because its utility in this area is unproven. We developed a new complementation-based reporter gene system where the large component of split NanoLuc luciferase (LgBiT) presented on the surface of cells (TM-LgBiT) interacts with a radiotracer consisting of the high-affinity complementary HiBiT peptide labeled with a radionuclide. Radiotracer uptake could be imaged in mice using SPECT/CT and bioluminescence within two hours of implanting reporter-gene-expressing cells. Imaging data were validated by ex vivo biodistribution studies. Following the demonstration of complementation between the TM-LgBiT protein and HiBiT radiotracer, we validated the use of the technology in the highly specific in vivo multimodal imaging of cells. These findings highlight the potential of this new approach to facilitate the advancement of cell and gene therapies from bench to clinic.

BackgroundFibroblast activation protein (FAP) is an attractive target for cancer theranostics. Although FAP-targeted nuclear imaging demonstrated promising clinical results, only sub-optimal results are reported for targeted radionuclide therapy (TRT). Preclinical research is crucial in selecting promising FAP-targeted radiopharmaceuticals and for obtaining an increased understanding of factors essential for FAP-TRT improvement. FAP is mainly expressed by cancer-associated fibroblasts in the tumor stroma and less on cancer cells themselves. Therefore, other (complex) factors impact FAP-TRT efficacy compared to currently clinically applied TRT strategies. For accurate evaluation of these aspects, selection of a representative preclinical model is important. Currently mainly human cancer cell lines transduced to (over)express FAP are applied, lacking clinical representation. It is unclear how these and more physiological FAP-expressing models compare to each other, and whether/how the model influences the study outcome. We aimed to address this by comparing FAP tracer behavior in FAP-transduced HT1080-huFAP and HEK293-huFAP cells, and endogenous FAP-expressing U-87 MG cancer cells and PS-1 pancreatic stellate cells. [111In]In-FAPI-46 and a fluorescent FAP-targeted tracer (RTX-1370S) were used to compare tracer binding/uptake and localization in vitro and ex vivo. Additionally, FAP expression was determined with RT-qPCR and anti-FAP IHC.ResultsAlthough FAP expression was highest in HEK293-huFAP cells and cell line derived xenografts, this did not result in the highest tracer uptake. [111In]In-FAPI-46 uptake was highest in HT1080-huFAP, closely followed by HEK293-huFAP, and a 6-10-fold lower uptake for U-87 MG and PS-1 cells. However, ex vivo U-87 MG xenografts only showed a 2-fold lower binding compared to HT1080-huFAP and HEK293-huFAP xenografts, mainly because the cell line attracts murine fibroblasts as demonstrated in our RT-qPCR and IHC studies.ConclusionsThe interaction between FAP and FAP-targeted tracers differs between models, indicating the need for appropriate model selection and that comparing results across studies using different models is difficult.

Each tumor has its own distinctive molecular identity. Treatment, therefore, should be tailored to this unique cancer phenotype. Theragnostics uses the same compound for targeted imaging and treatment, radiolabeled to an appropriate radionuclide, respectively. Gastrin-releasing peptide receptors (GRPRs) are overexpressed in prostate cancer, and radiolabeled GRPR antagonists have shown high diagnostic performance at staging and biochemical recurrence. Several GRPR-targeting theragnostic compounds have been developed preclinically. Their translation into clinics is underway with 4 clinical trials recruiting participants with GRPR-expressing tumors.

Radiolabeled NeoB is a promising gastrin-releasing peptide receptor (GRPR)–targeting radiopharmaceutical for theranostics of GRPR-expressing malignancies, e.g., prostate cancer (PCa). The aim of this study was to evaluate the effect of different doses of [177Lu]Lu-NeoB on the balance between therapeutic efficacy and safety in a preclinical PCa model. To determine the efficacy of [177Lu]Lu-NeoB, PC-3 xenografted mice received 3 sham injections (control group) or 3 injections of 30 MBq/300 pmol, 40 MBq/400 pmol, or 60 MBq/600 pmol [177Lu]Lu-NeoB (groups 1, 2, and 3, respectively) 1 week apart. To quantify tumor uptake, single-photon emission computed tomography/computed tomography (SPECT/CT) imaging was performed 4 h after the first, second, and third injection on a separate group of animals. For safety evaluations, pancreatic and renal tissues of non-tumor-bearing mice treated with the abovementioned [177Lu]Lu-NeoB doses were evaluated 12 and 24 weeks post-treatment. Treatment of PC-3 tumors with all three studied [177Lu]Lu-NeoB doses was effective. Median survival times were significantly (p < 0.0001) improved for treatment groups 1, 2, and 3 versus the control group (82 days, 89 days, 99 days versus 19 days, respectively). However, no significant differences were observed between treatment groups. Quantification of SPECT/CT images showed minimal differences in the average absolute radioactivity uptake, especially after the third injection. Histopathological analysis revealed no clear signs of treatment-related pancreatic toxicity. For the kidneys, atrophy and fibrosis were observed for one animal from group 1 and a chronic inflammatory response was observed for both animals from group 3 at 24 weeks post-treatment. Treatment with [177Lu]Lu-NeoB is effective in a preclinical PCa model. Adjusting the administered dose could positively impact the risk-benefit balance as a higher dose might not lead to an increased therapeutic effect, but it may lead to an increase in toxicological effects in healthy organs such as the kidneys.

Small-molecule drug conjugates (SMDCs) are compounds in which a therapeutic payload is conjugated to a targeting vector, for specific delivery to the tumor site. This promising approach can be translated to the treatment of prostate cancer by selecting a targeting vector which binds to the prostate-specific membrane antigen (PSMA). Moreover, the addition of a bifunctional chelator to the molecule allows for the use of both diagnostic and therapeutic radionuclides. In this way, the distribution of the SMDC in the body can be monitored, and combination therapy regimes can be implemented. We combined a glutamate-urea-lysine vector to the cytotoxic agent DM1 and a DOTA chelator via an optimized linker to obtain the theranostic SMDC (T-SMDC) ePSMA-DM1. ePSMA-DM1 retained a high binding affinity to PSMA and demonstrated PSMA-specific uptake in cells. Glutathione stability assays showed that the half-life of the T-SMDC in a reducing environment was 2 h, and full drug release was obtained after 6 h. Moreover, 100 nM of ePSMA-DM1 reduced the cell viability of the human PSMA-positive LS174T cells by >85% after 72 h of incubation, which was comparable to a 10-fold higher dose of free DM1. [111In]In-ePSMA-DM1 and [177Lu]Lu-ePSMA-DM1 were both obtained in high radiochemical yields and purities (>95%), with >90% stability in PBS and >80% stability in mouse serum for up to 24 h post incubation at 37 °C. SPECT/CT imaging studies allowed for a faint tumor visualization of [111In]In-ePSMA-DM1 at 1 h p.i., and the ex vivo biodistribution showed tumor uptake (2.39 ± 0.29% ID/g) at 1 h p.i., with the compound retained in the tumor for up to 24 h. Therefore, ePSMA-DM1 is a promising T-SMDC candidate for prostate cancer, and the data obtained so far warrant further investigations, such as therapeutic experiments, after further optimization.