Coaxial electrospinning of PEEU/gelatin to fiber meshes with enhanced mesenchymal stem cell attachment and proliferation.

Microfibers with a core-shell structure can be produced by co-axial electrospinning, allowing for the functionalization of the outer layer with bioactive molecules. In this study, a thermoplastic, degradable polyesteretherurethane (PEEU), consisting of poly(p-dioxanone) (PPDO) and poly(ɛ-caprolactone) (PCL) segments with different PPDO to PCL weight ratios, were processed into fiber meshes by co-axial electrospinning with gelatin. The prepared PEEU fibers have a diameter of 1.3±0.5 µm and an elastic modulus of around 5.1±1.0 MPa as measured by tensile testing in a dry state at 37°C, while the PEEU/Gelatin core-shell fibers with a gelatin content of 12±6 wt% and a diameter of 1.5±0.5 µm possess an elastic modulus of 15.0±1.1 MPa in a dry state at 37 °C but as low as 0.7±0.7 MPa when hydrated at 37 °C. Co-axial electrospinning allowed for the homogeneous distribution of the gelatin shell along the whole microfiber. Gelatin with conjugated Fluorescein (FITC) remained stable on the PEEU fibers after 7 days incubation in Phosphate-buffered saline (PBS) at 37 °C. The gelatin coating on PEEU fibers lead to enhanced human adipose tissue derived mesenchymal stem cell (hADSC) attachment and a proliferation rate 81.7±34.1 % higher in cell number in PEEU50/Gelatin fibers after 7 days of cell culture when compared to PEEU fibers without coating. In this work, we demonstrate that water-soluble gelatin can be incorporated as the outer shell of a polymer fiber via molecular entanglement, with a sustained presence and role in enhancing stem cell attachment and proliferation.

Elasticity of fiber meshes from multiblock copolymers influences endothelial cell behavior.

BACKGROUND: The behavior of endothelial cells is remarkably influenced by the physical and biochemical signals from their surrounding microenvironments. OBJECTIVE: Here, the elasticity of fiber meshes was studied as a design parameter of substrates for endothelial cells in order to modulate angiogenesis. METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured on electrospun fiber meshes made from polyetheresterurethane (PEEU), differing in their elasticity. Cell morphology, proliferation, migration and angiogenesis of endothelial cells on the degradable substrate meshes were characterized. RESULTS: The aspect ratio of HUVECs cultured on the fiber meshes from PEEU materials increased with increasing stiffness of the materials. HUVECs cultured on fiber meshes with high stiffness (Young's modulus E = 4.5±0.8 MPa) presented a higher proliferation rate and significantly faster migration velocity, as well as higher tube formation capability than the cells cultured on fiber meshes with low stiffness (E = 2.6±0.8 MPa). CONCLUSIONS: These results suggested that tuning the fiber meshes' elasticity might be a potential strategy for modulating the formation or regeneration of blood vessels.

The effect of stiffness variation of electrospun fiber meshes of multiblock copolymers on the osteogenic differentiation of human mesenchymal stem cells.

Electrospinning has attracted significant attention as a method to produce cell culture substrates whose fibrous structure mimics the native extracellular matrix (ECM). In this study, the influence of E-modulus of fibrous substrates on the lineage commitment of human adipose-derived stem cells (hADSCs) was studied using fiber meshes prepared via the electrospinning of a polyetheresterurethane (PEEU) consisting of poly(ρ-dioxanone) (PPDO) and poly(ɛ-caprolactone) (PCL) segments. The PPDO: PCL weight ratio was varied from 40:60 to 70:30 to adjust the physiochemical properties of the PEEU fibers. The cells attached on stiffer PEEU70 (PPDO:PCL,= 70:30) fiber meshes displayed an elongated morphology compared to those cultured on softer fibers. The nuclear aspect ratio (width vs. length of a nucleus) of hADSCs cultured on softer PEEU40 (PPDO:PCL = 40:60) fibers was lower than on stiffer fibers. The osteogenic differentiation of hADSCs was enhanced by culturing on stiffer fibers. Compared to PEEU40, a 73% increase of osteocalcin expression and a 34% enhancement of alkaline phosphatase (ALP) activity was observed in cells on PEEU70. These results demonstrated that the differentiation commitment of stem cells could be regulated via tailoring the mechanical properties of electrospun fibers.

Adipogenic differentiation of human adipose derived mesenchymal stem cells in 3D architectured gelatin based hydrogels (ArcGel).

Polymeric matrices mimicking multiple functions of the ECM are expected to enable a material induced regeneration of tissues. Here, we investigated the adipogenic differentiation of human adipose derived mesenchymal stem cells (hADSCs) in a 3D architectured gelatin based hydrogel (ArcGel) prepared from gelatin and L-lysine diisocyanate ethyl ester (LDI) in an one-step process, in which the formation of an open porous morphology and the chemical network formation were integrated. The ArcGel was designed to support adipose tissue regeneration with its 3D porous structure, high cell biocompatibility, and mechanical properties compatible with human subcutaneous adipose tissue. The ArcGel could support initial cell adhesion and survival of hADSCs. Under static culture condition, the cells could migrate into the inner part of the scaffold with a depth of 840±120 µm after 4 days, and distributed in the whole scaffold (2 mm in thickness) within 14 days. The cells proliferated in the scaffold and the fold increase of cell number after 7 days of culture was 2.55±0.08. The apoptotic rate of hADSCs in the scaffold was similar to that of cells maintained on tissue culture plates. When cultured in adipogenic induction medium, the hADSCs in the scaffold differentiated into adipocytes with a high efficiency (93±1%). Conclusively, this gelatin based 3D scaffold presented high cell compatibility for hADSC cultivation and differentiation, which could serve as a potential implant material in clinical applications for adipose tissue reparation and regeneration.

Modulation of the mesenchymal stem cell migration capacity via preconditioning with topographic microstructure.

Controlling mesenchymal stem cells (MSCs) behavior is necessary to fully exploit their therapeutic potential. Various approaches are employed to effectively influence the migration capacity of MSCs. Here, topographic microstructures with different microscale roughness were created on polystyrene (PS) culture vessel surfaces as a feasible physical preconditioning strategy to modulate MSC migration. By analyzing trajectories of cells migrating after reseeding, we demonstrated that the mobilization velocity of human adipose derived mesenchymal stem cells (hADSCs) could be promoted by and persisted after brief preconditioning with the appropriate microtopography. Moreover, the elevated activation levels of focal adhesion kinase (FAK) and mitogen-activated protein kinase (MAPK) in hADSCs were also observed during and after the preconditioning process. These findings underline the potential enhancement of in vivo therapeutic efficacy in regenerative medicine via transplantation of topographic microstructure preconditioned stem cells.

Influence of surface roughness on neural differentiation of human induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) own the capacity to develop into all cell types of the adult body, presenting high potential in regenerative medicine. Regulating and controlling the differentiation of iPSCs using the surface topographic cues of biomaterials is a promising and safe approach to enhance their therapeutic efficacy. In this study, we tested the effects of surface roughness on differentiation of human iPSCs into neural progenitor cells and dopaminergic neuron cells using polystyrene with different roughness (R0: flat surface; R1: rough surface, Rq ∼ 6 µm; R2: rough surface, Rq ∼ 38 µm). Neural differentiation of human iPSCs could be influenced by surface roughness. Up-regulated neuronal markers were found in cells on rough surface, as examined by real-time PCR and immunostaining. Particularly, the R1 surface significantly improved the neuronal marker expression, as compared to R0 and R2 surface. This study demonstrates the significance of surface roughness, depending on the roughness level, in promoting differentiation of human iPSCs towards the neuronal lineage. Our study suggests the potential applications of surface roughness in iPSCs based treatment of neural disorder diseases, and highlights the importance of design and development of biomaterials with effective surface structures to regulate stem cells.

Molecular Imaging of Post-Src Inhibition Tumor Signatures for Guiding Dasatinib Combination Therapy.

UNLABELLED: Noninvasive, real-time, quantitative measurement of key biomarkers associated with cancer therapeutic interventions could provide a better understanding of cancer biology. We investigated in this study whether incorporating multiple molecular imaging approaches could be used to guide dasatinib anti-Src therapy and aid in the rational design of a combination therapy regimen. METHODS: Bioluminescence imaging, (18)F-FDG PET, integrin αvß3-targeted SPECT/CT, and vascular endothelial growth factor-targeted near-infrared fluorescence imaging were performed before and after dasatinib treatment in a tumor mouse model. RESULTS: There was no significant difference in the bioluminescence imaging signal or (18)F-FDG tumor uptake in dasatinib-treated tumors compared with the control tumors. However, the uptake of (99m)T-3PRGD2 (integrin αvß3-specific) and DyLight755-ranibizumab (vascular endothelial growth factor-specific) in the dasatinib-treated tumors was significantly lower than that in the control tumors. In vitro studies confirmed the antiangiogenic effects of dasatinib but indicated a lack of cytotoxicity. Dasatinib plus cytotoxic docetaxel elicited marked synergistic tumor growth inhibition in vivo. CONCLUSION: Visualization of post-Src inhibition tumor signatures through multiple imaging approaches facilitates sensitive and quantitative measurement of cancer biomarkers in vivo, thus aiding in the rational design of dasatinib combination therapy.

Small-Animal SPECT/CT of the Progression and Recovery of Rat Liver Fibrosis by Using an Integrin αvß3-targeting Radiotracer.

PURPOSE: To assess the potential utility of an integrin αvß3-targeting radiotracer, technetium 99m-PEG4-E[PEG4-cyclo(arginine-glycine-aspartic acid-D-phenylalanine-lysine)]2 ((99m)Tc-3PRGD2), for single photon emission computed tomography (SPECT)/computed tomography (CT) for monitoring of the progression and prognosis of liver fibrosis in a rat model. MATERIALS AND METHODS: All animal experiments were performed by following the protocol approved by the institutional animal care and use committee. (99m)Tc-3PRGD2 was prepared and longitudinal SPECT/CT was performed to monitor the progression (n = 8) and recovery (n = 5) of liver fibrosis induced in a rat model by means of thioacetamide (TAA) administration. The mean liver-to-background radioactivity per unit volume ratio was analyzed for comparisons between the TAA and control (saline) groups at different stages of liver fibrosis. Data were compared by using Student t and Mann-Whitney tests. Results:of SPECT/CT were compared with those of ex vivo biodistribution analysis (n = 5). RESULTS: Accumulation of (99m)Tc-3PRGD2 in the liver increased in proportion to the progression of fibrosis and TAA exposure time; accumulation levels were significantly different between the TAA and control groups as early as week 4 of TAA administration (liver-to-background ratio: 32.30 ± 3.39 vs 19.01 ± 3.31; P = .0002). Results of ex vivo immunofluorescence staining demonstrated the positive expression of integrin αvß3 on the activated hepatic stellate cells, and the integrin αvß3 levels in the liver corresponded to the results of SPECT/CT (R(2) = 0.75, P < .0001). (99m)Tc-3PRGD2 uptake in the fibrotic liver decreased after antifibrotic therapy with interferon α2b compared with that in the control group (relative liver-to-background ratio: 0.45 ± 0.05 vs 1.01 ± 0.05; P < .0001) or spontaneous recovery (relative liver-to-background ratio: 0.56 ± 0.06 vs 1.01 ± 0.05; P < .0001). CONCLUSION: (99m)Tc-3PRGD2 SPECT/CT was successfully used to monitor the progression and recovery of liver fibrosis and shows potential applications for noninvasive diagnosis of early stage liver fibrosis.

Recent Advances in Molecular Image-Guided Cancer Radionuclide Therapy.

Cancer-targeted radionuclide therapy is a promising approach for the treatment of a wide variety of malignancies, especially those resistant to conventional therapies. However, to improve the use of targeted radionuclide therapy for the management of cancer patients, the in vivo behaviors, dosimetry, and efficacy of radiotherapeutic agents need to be well characterized and monitored. Molecular imaging, which is a powerful tool for the noninvasive characterization and quantification of biological processes in living subjects at the cellular and molecular levels, plays an important role in the guidance of cancer radionuclide therapy. In this review, we introduce the radiotherapeutics for cancer-targeted therapy and summarize the most recent evidence supporting the use of molecular imaging to guide cancer radionuclide therapy.

Molecular imaging of tumor-infiltrating macrophages in a preclinical mouse model of breast cancer.

Significant evidence has indicated that tumor-associated macrophages (TAMs) play a critical role in the proliferation, invasion, angiogenesis, and metastasis of a variety of human carcinomas. In this study, we investigated whether near-infrared fluorescence (NIRF) imaging using a macrophage mannose receptor (MMR; CD206)-targeting agent could be used to noninvasively visualize and quantify changes in TAMs in vivo. The CD206-targeting NIRF agent, Dye-anti-CD206, was prepared and characterized in vitro and in vivo. By using NIRF imaging, we were able to noninvasively image tumor-infiltrating macrophages in the 4T1 mouse breast cancer model. Importantly, longitudinal NIRF imaging revealed the depletion of macrophages in response to zoledronic acid (ZA) treatment. However, ZA alone did not lead to the inhibition of 4T1 tumor growth. We therefore combined anti-macrophage ZA therapy and tumor cytotoxic docetaxel (DTX) therapy in the mouse model. The results demonstrated that this combination strategy could significantly inhibit tumor growth as well as tumor metastasis to the lungs. Based on these findings, we concluded that CD206-targeted molecular imaging can sensitively detect the dynamic changes in tumor-infiltrating macrophages, and that the combination of macrophage depletion and cytotoxic therapy is a promising strategy for the effective treatment of solid tumors.

Serial in vivo imaging using a fluorescence probe allows identification of tumor early response to cetuximab immunotherapy.

Cetuximab is an antiepidermal growth factor receptor (EGFR) monoclonal antibody that has received the approval of the Food and Drug Administration (FDA) for cancer treatment. However, most clinical studies indicate that cetuximab can only elicit positive effects on a subset of cancer patients. In this study, we investigated whether near-infrared fluorescence (NIRF) imaging of tumor vascular endothelial growth factor (VEGF) expression could be a biomarker for tumor early response to cetuximab therapy in preclinical wild-type and mutant tumor models of the KRAS gene. The treatment efficacy of cetuximab was determined in both HT-29 (wild-type KRAS) and HTC-116 (mutant KRAS) human colon cancer models. A VEGF-specific optical imaging probe (Dye755-Ran) was synthesized by conjugating ranibizumab (an anti-VEGF antibody Fab fragment) with a NIRF dye. Serial optical scans with Dye755-Ran were performed in HT-29 and HTC-116 xenograft models. By using longitudinal NIRF imaging, we were able to detect early tumor response on day 3 and day 5 after initiation of cetuximab treatment in the cetuximab-responsive HT-29 tumor model. Enzyme-linked immunosorbent assay (ELISA) confirmed that cetuximab treatment inhibited human VEGF expression in the KRAS wild-type HT-29 tumor but not in the KRAS mutant HCT-116 tumor. We have demonstrated that the antitumor effect of cetuximab can be noninvasively monitored by serial fluorescence imaging using Dye755-Ran. VEGF expression detected by optical imaging could serve as a sensitive biomarker for tumor early response to drugs that directly or indirectly act on VEGF.

Integrin αvß6-Targeted SPECT Imaging for Pancreatic Cancer Detection.

UNLABELLED: Integrin αvß6, a member of the integrin family, is specifically expressed in many malignancies but not in normal organs. Overexpression of integrin αvß6 is usually correlated with malignant potential and poor prognosis. In this study, we describe the synthesis and evaluation of a (99m)Tc-labeled integrin αvß6-targeting peptide as a SPECT radiotracer for the in vivo imaging of integrin αvß6 expression. METHODS: An integrin αvß6-targeting peptide (denoted as the HK peptide) was conjugated with 6-hydrazinonicotinyl (HYNIC) and radiolabeled with (99m)Tc using tricine and TPPTS (trisodium triphenylphosphine-3,3',3″-trisulfonate) as coligands. The in vitro and in vivo characteristics of (99m)Tc-HYNIC(tricine)(TPPTS)-HK ((99m)Tc-HHK) were investigated in BxPC-3 (integrin αvß6-positive) and HEK293 (integrin αvß6-negative) models. The ability of (99m)Tc-HHK to detect liver metastasis of pancreatic cancer was evaluated using small-animal SPECT/CT. RESULTS: (99m)Tc-HHK showed high integrin αvß6-binding specificity both in vitro and in vivo. (99m)Tc-HHK was cleared rapidly from the blood and normal organs except for the kidneys. The highest uptake (0.88 ± 0.12 percentage injected dose per gram) of (99m)Tc-HHK in BxPC-3 tumors was observed at 0.5 h after injection. High-contrast images of integrin αvß6-positive tumors were obtained using (99m)Tc-HHK. The minimum nonspecific activity accumulation in normal liver tissues rendered high-quality SPECT/CT images of metastatic lesions. CONCLUSION: (99m)Tc-HHK is a promising SPECT radiotracer for the noninvasive imaging of integrin αvß6 expression in vivo. SPECT/CT with (99m)Tc-HHK could provide an effective approach for the noninvasive detection of primary and metastatic lesions of integrin αvß6-positive tumors.

Molecular imaging reveals trastuzumab-induced epidermal growth factor receptor downregulation in vivo.

UNLABELLED: Previous in vitro studies demonstrated that treating tumors expressing both epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 with trastuzumab resulted in increased EGFR homodimerization and subsequent rapid downregulation of EGFR. We investigated whether molecular imaging using near-infrared fluorescence (NIRF) imaging and PET probes could sensitively detect trastuzumab-induced EGFR downregulation in vivo. METHODS: The F(ab')2 antibody fragment PaniF(ab')2 was generated by digesting the anti-EGFR monoclonal antibody panitumumab. PaniF(ab')2 was labeled with either a NIRF dye or (68)Ga, and optical imaging and small-animal PET imaging of Dye-PaniF(ab')2 and (68)Ga-PaniF(ab')2, respectively, were performed in HT-29 tumor-bearing nude mice treated with trastuzumab or untreated control. RESULTS: Longitudinal NIRF imaging studies revealed significantly reduced tumor uptake of Dye-PaniF(ab')2 on days 5 and 7 in trastuzumab-treated HT-29 tumors, compared with control. Western blotting confirmed the downregulation of EGFR after treatment with trastuzumab. Small-animal PET on day 5 after trastuzumab treatment also demonstrated decreased (68)Ga-PaniF(ab')2 uptake in trastuzumab-treated HT-29 tumors. The tumor uptake value of (68)Ga-PaniF(ab')2 obtained from PET imaging had an excellent linear correlation with the uptake value measured using biodistribution. CONCLUSION: The downregulation of EGFR induced by trastuzumab treatment could be detected noninvasively using optical and PET imaging. This molecular imaging strategy could provide a dynamic readout of changes in the tumor signaling and may facilitate the noninvasive monitoring of the early tumor response to drug treatment.

Early assessment of tumor response to gefitinib treatment by noninvasive optical imaging of tumor vascular endothelial growth factor expression in animal models.

UNLABELLED: Epidermal growth factor receptor (EGFR) expression is upregulated in many types of tumors, and the EGFR tyrosine kinase inhibitor gefitinib has high potential as an anticancer drug. However, accumulating clinical evidence has indicated that only a subset of patients benefit from gefitinib treatment. This study aimed to determine whether optical imaging of vascular endothelial growth factor (VEGF) expression can be an early biomarker for tumor response to gefitinib therapy. METHODS: A VEGF-targeting fluorescent probe Dye-BevF(ab')2 was prepared and tested in vivo. Longitudinal optical imaging studies using Dye-BevF(ab')2 were performed in both 22B (gefitinib-resistant) and A549 (gefitinib-responsive) tumor models at different times (days 0, 2, and 5) before and after gefitinib treatment. The imaging results were validated by ex vivo immunofluorescence staining and enzyme-linked immunosorbent assay. RESULTS: Dye-BevF(ab')2 exhibited high specificity for VEGF in vivo. There was no significant change in the Dye-BevF(ab')2 uptake in gefitinib-treated 22B tumors, compared with the control group. In contrast, the A549 tumor uptake of Dye-BevF(ab')2 in the gefitinib-treated group was significantly lower on days 2 and 5 than that in the control group and at the baseline. An in vivo gefitinib treatment study confirmed that 22B tumors were gefitinib-resistant, whereas A549 tumors were gefitinib-responsive. Immunofluorescence staining and enzyme-linked immunosorbent assay confirmed that changes in the Dye-BevF(ab')2 uptake were correlated with VEGF expression levels in tumors. CONCLUSION: Optical imaging of VEGF expression with Dye-BevF(ab')2 can be used for the early assessment of tumor response to gefitinib therapy. This approach may also be valuable for preclinical high-throughput screening of novel antiangiogenic drugs.

Longitudinal monitoring of tumor antiangiogenic therapy with near-infrared fluorophore-labeled agents targeted to integrin αvß3 and vascular endothelial growth factor.

PURPOSE: Optical imaging is emerging as a powerful tool for the noninvasive imaging of the biological processes in living subjects. This study aimed to investigate whether optical imaging of integrin αvß3 and vascular endothelial growth factor (VEGF) expression can serve as sensitive biomarkers for tumor early response to antiangiogenic therapy. METHODS: We synthesized two near-infrared fluorescence (NIRF) imaging agents, CF680R-3PRGD2 and CF750-BevF(ab')2, which were designed to specifically bind to integrin αvß3 and VEGF, respectively. The ability of optical imaging using the two imaging agents for early monitoring the antiangiogenic effect of sunitinib was evaluated. RESULTS: CF680R-3PRGD2 and CF750-BevF(ab')2 specifically bound to their respective targets in vitro and in HT-29 tumor-bearing nude mice. Sunitinib treatment led to significantly decreased tumor uptake of CF680R-3PRGD2 (e.g., 7.47 ± 1.62 % vs. 4.24 ± 0.16 % on day 4; P < 0.05) and CF750-BevF(ab')2 (e.g., 7.43 ± 2.43 % vs. 4.04 ± 1.39 % on day 2; P < 0.05) in vivo. Immunofluorescence staining and an enzyme-linked immunosorbent assay confirmed that sunitinib-induced changes in tumor uptake of CF680R-3PRGD2 and CF750-BevF(ab')2 were correlated with changes in the levels of integrin αvß3 and VEGF. Radiobiodistribution of (99m)Tc-3PRGD2 and (125)I-BevF(ab')2, the radiocounterparts of CF680R-3PRGD2 and CF750-BevF(ab')2, respectively, also validated optical imaging results. CONCLUSION: Longitudinal monitoring of tumor integrin αvß3 and VEGF expression could be used as early biomarkers for tumor response to antiangiogenic therapy. This strategy may facilitate the development of new antiangiogenic drugs, and be used for elucidation of the underlying mechanisms of therapies involving the integrin and the VEGF signaling pathway.

177Lu-labeled antibodies for EGFR-targeted SPECT/CT imaging and radioimmunotherapy in a preclinical head and neck carcinoma model.

Epidermal growth factor receptor (EGFR) has been well characterized as an important target for cancer therapy. Immunotherapy based on EGFR-specific antibodies (e.g., panitumumab and cetuximab) has shown great clinical promise. However, increasing evidence has indicated that only a subgroup of patients receiving these antibodies will benefit from them, and even patients who do initially experience a major response may eventually develop therapeutic resistance. In this study, we investigated whether panitumumab and cetuximab can serve as delivery vehicles for tumor-targeted radionuclide therapy in a preclinical tumor model that did not respond to immunotherapy. The in vitro toxicity and cell binding properties of panitumumab and cetuximab were characterized. Both antibodies were conjugated with 1,4,7,10-tetraazadodecane-N,N',N",N"'-tetraacetic acid (DOTA) and radiolabeled with (177)Lu. Small-animal SPECT/CT, biodistribution, and radioimmunotherapy (RIT) studies of (177)Lu-DOTA-panitumumab ((177)Lu-Pan) and (177)Lu-DOTA-cetuximab ((177)Lu-Cet) were performed in the UM-SCC-22B tumor model. Both (177)Lu-Pan and (177)Lu-Cet exhibited favorable tumor targeting efficacy. The tumor uptake was 20.92 ± 4.45, 26.10 ± 5.18, and 13.27 ± 1.94% ID/g for (177)Lu-Pan, and 15.67 ± 3.84, 15.72 ± 3.49, and 7.82 ± 2.36% ID/g for (177)Lu-Cet at 24, 72, and 120 h p.i., respectively. RIT with a single dose of 14.8 MBq of (177)Lu-Pan or (177)Lu-Cet significantly delayed tumor growth. (177)Lu-Pan induced more effective tumor growth inhibition due to a higher tumor uptake. Our results suggest that panitumumab and cetuximab can function as effective carriers for tumor-targeted delivery of radiation, and that RIT is promising for targeted therapy of EGFR-positive tumors, especially for those tumors that are resistant to antibody-based immunotherapy.