Locally advanced pancreatic tumours are highly resistant to conventional radiochemotherapy. Here we show that such resistance can be surmounted by an injectable depot of thermally responsive elastin-like polypeptide (ELP) conjugated with iodine-131 radionuclides (131I-ELP) when combined with systemically delivered nanoparticle albumin-bound paclitaxel. This combination therapy induced complete tumour regressions in diverse subcutaneous and orthotopic mouse models of locoregional pancreatic tumours. 131I-ELP brachytherapy was effective independently of the paclitaxel formulation and dose, but external beam radiotherapy (EBRT) only achieved tumour-growth inhibition when co-administered with nanoparticle paclitaxel. Histological analyses revealed that 131I-ELP brachytherapy led to changes in the expression of intercellular collagen and junctional proteins within the tumour microenvironment. These changes, which differed from those of EBRT-treated tumours, correlated with the improved delivery and accumulation of paclitaxel nanoparticles within the tumour. Our findings support the further translational development of 131I-ELP depots for the synergistic treatment of localized pancreatic cancer.
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The main data supporting the results in this study are available within the paper and its Supplementary Information. The raw and analysed datasets generated during the study are available for research purposes from the corresponding author on reasonable request. Source data are provided with this paper.
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We thank Z. Su of the Duke Pathology Department for invaluable assistance in processing and assessing the histological specimens; L. d. S. Campos for expertise in operating the X-RAD CX225 micro-irradiator; and S. Kron of the University of Chicago for early discussions that inspired our investigation of vascular permeability following radiotherapy. The following Duke Shared Facilities provided resources critical to this study: the Duke Cancer Center Isolation Facility for animal housing and husbandry, the DCI Optical Molecular Imaging and Analysis Shared Resource for in vivo imaging methods, the Duke Research Immunohistology Laboratory for tissue pathology, and the Duke Light Microscopy Core Facility. Funding for these studies was provided by the NIH through grant 5R01EB000188 to A.C. and NCI grant R35CA197616 to D.G.K.
A.C. is a scientific advisor to PhaseBio Pharmaceuticals, which has licensed the ELP technology for drug delivery from Duke University. D.G.K. is a co-founder of XRAD Therapeutics, which develops small-molecule radiosensitizers, and is on the Scientific Advisory Board of Lumicell, Inc., which commercializes intraoperative imaging technology. A provisional patent application (US20220008567A1, United States, 2018) has been filed by Duke University on the basis of this work, with J.L.S., A.C. and W.L. listed as inventors.
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Single agent therapy data from the 131I-ELP dose escalation trial. (a) 3.3 µCi/mm3 treatment group showed no improvement over untreated tumors (p > 0.05, 2-way ANOVA), nor (b) was there any significant survival advantage (p > 0.05, Mantel Cox log-rank). (c) The 6.6 µCi/mm3 treatment group showed improved single agent efficacy (p = 0.047, 2-way ANOVA) but combination therapy was still superior (p = 0.037, 2-way ANOVA). (d) Median survival for the 6.6 µCi/mm3 monotherapy group did increase to 33d, compared to 24d and 19d for the 3.3 µCi/mm3 and PBS control groups, respectively (p = 0.005, Mantel Cox log-rank). (e) The 10.0 µCi/mm3 treatment group exhibited improved tumor regression trends, but benefits were insignificant compared with previous 131I-ELP doses (p = 0.762, 2-way ANOVA). (f) As a monotherapy, the 10.0 µCi/mm3 monotherapy provided significant benefit over CP-PTX only and untreated tumors (p = 0.005), but the combination group significantly outperformed the monotherapy (p < 0.004, Mantel Cox log-rank).
Single agent therapy data from CP-PTX dose escalation study. (a) Tumor response to for the single agent CP-PTX group dosed at 50 mg/kg was insignificant compared to PBS control mice (p = 0.063, 2-way ANOVA) (b) 50 mg/kg CP-PTX also conferred no survival advantage over unless combined with 131I-ELP (p = 0.333 Mantel Cox log-rank). (c) The 25 mg/kg treatment group also demonstrated no improvement in tumor response compared to untreated tumors (p = 0.129, 2-way ANOVA). (d) The 25 mg/kg group median survival was 25d compared to 21d for the PBS controls (p = 0.127, Mantel Cox log-rank). (e) Similarly, the 12.5 mg/kg CP-PTX group showed no improvement in tumor response and (f) had an identical median survival of 21d as PBS treated control mice. At all doses, CP-PTX required the combination of 131I-ELP to significantly improve tumor responses.
Bliss Independence isobolograms were constructed to evaluate if combination therapy responses were synergistic or merely the result of additive effects. Trials analyzed included: (a) 131I-ELP with single i.v. dose of CP-PTX, (b) 131I-ELP with two i.v. doses of CP-PTX, (c) 131I-ELP with four i.v. doses of nab-paclitaxel, (d) 10 Gy x5 fractions EBRT with four i.v. doses of CP-PTX., (e) 5 Gy x5 fraction EBRT with four i.v. doses of CP-PTX, and (f) 3 Gy x15 fractions EBRT with four i.v. doses of CP-PTX. Mathematical synergy is indicated when the actual tumor regression (solid line) is lower than the Bliss prediction (dashed line) and exceeds the 95% confidence interval (shaded). All data are shown as mean ± SEM, with significance determined using 2-way ANOVA.
Hematoxylin and eosin histology images of treated tumor specimens representing (a) normal murine pancreas, (b) untreated BxPc3-luc2 tumors, (c) CP-PTX treated tumors, (d) 25 Gy EBRT monotherapy, (e) 131I-ELP monotherapy, (f) EBRT combination therapy, and (g) 131I-ELP combination therapy treated tumors. Full panels show representative pathological patterning, while insets emphasize cellular characteristics.
Masson Trichrome staining of treated tumors for detection of stromal collagen (cyan), cytoplasm (pink), and nuclei (purple). Tissue samples represented are taken from (a) normal murine pancreatic tissue and (b) an untreated s.c. BxPc3-luc2 tumor xenograft specimen, and (c) an orthotopic BxPc3-luc2 pancreatic tumor. Stromal differentiation was analyzed following tumor treatment with (d) CP-PTX monotherapy, (e) EBRT monotherapy, (f) 131I-ELP monotherapy, (g) EBRT combination therapy, and (h) 131I-ELP combination therapy. Tumor samples were collected 12 days after treatment. Insets emphasize cellular features.
Extended Data Fig. 6 Pathological analysis of treated BxPc3-luc2 tumors with immunohistochemistry staining.
Pathological analysis of treated BxPc3-luc2 tumors after immunohistochemistry staining. (a) Qualitative analysis of tumor stromal collagen after Masson Trichrome staining. Relative abundance was ranked by abundance: 0-normal collagen, 1-minimal stroma, 2-light stroma, 3-moderate stroma, 4-dense stroma. EBRT was found to induce a dense phenotype while 131I-ELP combination therapy significantly reduced stromal collagen into the light-moderate range (p = 0.005, ANOVA). (b) Claudin-4 cell membrane intensity staining was quantified after cell membrane IHC staining: 3-intense, 2-moderate, 1-light, and 0-no staining. (c) The relative area of Claudin-4 coverage was quantified by converting to a binary mask. Significant (p = 0.005, ANOVA) reduction in staining was observed for CP-PTX, 131I-ELP monotherapy, and 131I-ELP combination therapy treatments. (d) CD-31 (PECAM-1) staining of tumor cell cytoplasm, as evaluated by a blinded qualitative intensity scaling, showed no differences between treatments. (e) The relative area of CD-31 staining coverage also showed comparable expression amongst treatment groups. (f) Tumor tissue was next issued a CD-144 (VE-Cadherin) histology score (H-Score) that combined the intensity and frequency of nuclear staining within cells. Results showed a significant reduction in CD-144 for 131I-ELP groups over other single monotherapies and untreated tumor specimens (p = 0.040, ANOVA). (g) Area of expression showed a trend of reduced VE-Cadherin expression for 131I-ELP treatment groups but was not statistically significant.
Claudin-4 immunohistochemistry images for treated tumors, including (a) a normal murine pancreatic tissue and (b) an untreated orthotopic BxPc3-luc2 tumor. Claudin-4 expression was then assessed after treatment with (c) CP-PTX only, (d) EBRT (25 Gy) only, (e) 131I-ELP only, (f) EBRT combination therapy with CP-PTX, and (g) 131I-ELP combination therapy.
CD-31 immunohistochemistry staining for treated tumor specimens, consisting of (a) a normal murine prostate and (b) an untreated BxPc3-luc2 tumor, (c) CP-PTX only, (d) EBRT (25 Gy) only, (e) 131I-ELP only, (f) EBRT combination therapy with CP-PTX, and (g) 131I-ELP combination therapy with CP-PTX. No significant difference in expression was observed amongst treatments.
CD-144 immunohistochemistry staining of VE-Cadherin for treated tumor specimens, consisting of (a) a normal murine prostate and (b) an untreated BxPc3-luc2 tumor, (c) CP-PTX only, (d) EBRT (25 Gy) only, (e) 131I-ELP only, (f) EBRT combination therapy with CP-PTX, and (g) 131I-ELP combination therapy.
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Schaal, J.L., Bhattacharyya, J., Brownstein, J. et al. Brachytherapy via a depot of biopolymer-bound 131I synergizes with nanoparticle paclitaxel in therapy-resistant pancreatic tumours. Nat. Biomed. Eng 6, 1148–1166 (2022). https://doi.org/10.1038/s41551-022-00949-4
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