Collagen type I PET/MRI enables evaluation of treatment response in pancreatic cancer in pre-clinical and first-in-human translational studies

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is an invasive and rapidly progressive malignancy. A major challenge in patient management is the lack of a reliable imaging tool to monitor tumor response to treatment. Tumor-associated fibrosis characterized by high type I collagen is a hallmark of PDAC, and fibrosis further increases in response to neoadjuvant chemoradiotherapy (CRT). We hypothesized that molecular positron emission tomography (PET) using a type I collagen-specific imaging probe, ⁶⁸Ga-CBP8 can detect and measure changes in tumor fibrosis in response to standard treatment in mouse models and patients with PDAC. Methods: We evaluated the specificity of ⁶⁸Ga-CBP8 PET to tumor collagen and its ability to differentiate responders from non-responders based on the dynamic changes of fibrosis in nude mouse models of human PDAC including FOLFIRNOX-sensitive (PANC-1 and PDAC6) and FOLFIRINOX-resistant (SU.86.86). Next, we demonstrated the specificity and sensitivity of ⁶⁸Ga-CBP8 to the deposited collagen in resected human PDAC and pancreas tissues. Eight male participant (49-65 y) with newly diagnosed PDAC underwent dynamic ⁶⁸Ga-CBP8 PET/MRI, and five underwent follow up ⁶⁸Ga-CBP8 PET/MRI after completing standard CRT. PET parameters were correlated with tumor collagen content and markers of response on histology. Results: ⁶⁸Ga-CBP8 showed specific binding to PDAC compared to non-binding ⁶⁸Ga-CNBP probe in two mouse models of PDAC using PET imaging and to resected human PDAC using autoradiography (P < 0.05 for all comparisons). ⁶⁸Ga-CBP8 PET showed 2-fold higher tumor signal in mouse models following FOLFIRINOX treatment in PANC-1 and PDAC6 models (P < 0.01), but no significant increase after treatment in FOLFIRINOX resistant SU.86.86 model. ⁶⁸Ga-CBP8 binding to resected human PDAC was significantly higher (P < 0.0001) in treated versus untreated tissue. PET/MRI of PDAC patients prior to CRT showed significantly higher ⁶⁸Ga-CBP8 uptake in tumor compared to pancreas (SUV_mean: 2.35 ± 0.36 vs. 1.99 ± 0.25, P = 0.036, n = 8). PET tumor values significantly increased following CRT compared to untreated tumors (SUV_mean: 2.83 ± 0.30 vs. 2.25 ± 0.41, P = 0.01, n = 5). Collagen deposition significantly increased in response to CRT (59 ± 9% vs. 30 ± 9%, P=0.0005 in treated vs. untreated tumors). Tumor and pancreas collagen content showed a positive direct correlation with SUV_mean (R² = 0.54, P = 0.0007). Conclusions: This study demonstrates the specificity of ⁶⁸Ga-CBP8 PET to tumor type I collagen and its ability to differentiate responders from non-responders based on the dynamic changes of fibrosis in PDAC. The results highlight the potential use of collagen PET as a non-invasive tool for monitoring response to treatment in patients with PDAC.

Keywords: PET imaging; fibrosis; pancreatic cancer; treatment response; type I collagen.

Figure 1

Collagen increases in the tumor extracellular matrix in response to chemotherapy in mouse models of human PDAC. (A) Cell viability assay at various concentrations of FOLFIRINOX chemotherapy demonstrating the sensitivity of PDAC6 and PANC-1 cells at 24- and 48-h post-treatment and the relative resistance of SU.86.86 cells under the same conditions (n = 3-6 wells/condition). (B) Representative histological evaluation of pancreas and subcutaneously grown tumor tissues in the nude mice extracted before and at multiple days after treatment with FOLFIRINOX (administered every 3 days) and stained with hematoxylin and eosin (H&E), Masson Trichrome and Picro-Sirius Red (PSR) for collagen, immunohistochemical staining for type I collagen, and cleaved caspase-3 for apoptosis (Scale bar: 200 µm). (C) Quantitative analysis of collagen from PSR stained tissue reported as collagen proportional area (CPA). (D) Measure of collagen on the extracted tumors reported as hydroxyproline-to-proline (Hyp/Pro) ratio. (E) Quantitative immunohistochemical staining of apoptosis marker cleaved caspase-3 in the tumors at multiple time points. Each data point represents one mouse. Data are shown in mean ± SD. P values are the result of one-way ANOVA with multiple comparisons and post hoc Tukey test.

Figure 2

⁶⁸Ga-CBP8 specifically binds to collagen type 1 in mouse models of PDAC. (A) Structure of the type I collagen binding ⁶⁸Ga-CBP8 and control nonbinding ⁶⁸Ga-CNBP probes. There are 3 NODAGA moieties for potential Ga-68 labeling and the radiolabeled product is a mixture of these three isomers with a representative isomer shown here. (B) Schematic illustration of the study design for testing the specificity of ⁶⁸Ga-CBP8 and control ⁶⁸Ga-CNBP probes in untreated PDAC models based on dynamic PET over two consecutive days. (C) Tumor uptake curves from dynamic PET acquired 0-60 min after intravenous injection of each probe (⁶⁸Ga-CBP8 in blue line and ⁶⁸Ga-CNBP in black line) quantified as the percentage of injected dosage per cc (%ID/cc) (n = 4-7 male nude mice, Data are shown in mean ± SD). (D) Clearance of the ⁶⁸Ga-CBP8 from the blood (red line) over 60 min and tumor-to-blood uptake ratio (green line) reaching above 1 at 30-60 min post-injection (n = 5-6, Data are shown in mean ± SD). (E) Representative Picro-Sirius Red stained tissue of extracted tumors and correlation of collagen proportional area (CPA) with the mean tumor PET uptake values at 30-60 min of imaging with the specific ⁶⁸Ga-CBP8 (R² = 0.67, P = 0.001) and linear control ⁶⁸Ga-CNBP (R² = 0.015, P = 0.72) (each data point represents one mouse). PANC-1 data is shown in orange and PDAC6 in blue color.

Figure 3

⁶⁸Ga-CBP8 shows specific changes in type I collagen in response to chemotherapy in mouse models of PDAC. (A) Schematic illustration of study design to demonstrate the specificity of ⁶⁸Ga-CBP8 compared to control ⁶⁸Ga-CNBP PET over multiple days of treatment with FOLFIRINOX (administered every 3 days) in PANC-1 tumor-bearing mice (male nude mice, n = 4-6/group). (B-C) Representative coronal PET images (tumors shown in yellow circle) and quantitative analyses demonstrating a significantly higher uptake of ⁶⁸Ga-CBP8 in the tumors at all time points compared to the control ⁶⁸Ga-CNBP (paired t-test, two-tailed), highlighting the specificity of the probe for collagen I. Data also show an increase in tumor ⁶⁸Ga-CBP8 following FOLFIRINOX treatment but no increase in ⁶⁸Ga-CNBP tumor uptake with treatment. Data are shown in mean ± SD.

Figure 4

⁶⁸Ga-CBP8 enables monitoring of the response to chemotherapy in mouse models of human PDAC and differentiates responders from non-responders. (A) Schematic study design. (B) Representative coronal PET images of the nude mice with PANC-1 and SU.86.86 tumors imaged from 30-60 min after injection of ⁶⁸Ga-CBP8 at days 0, 9, and 15 after treatment with intravenous FOLFIRINOX or vehicle (tumors shown in yellow circle). (C) Quantitative analyses of ⁶⁸Ga-CBP PET tumor uptake at days 0, 9, and 15 of treatment with FOLFIRINOX or vehicle (n = 4-8/group, ANOVA with multiple comparisons and post hoc Tukey test). (D) Tumor volume curves over 24 days of treatment with FOLFIRINOX or vehicle. Data are shown in mean ± SD.

Figure 5

⁶⁸Ga-CBP8 detects fibrosis and measures response to neoadjuvant chemoradiotherapy in resected human pancreatic tumor tissues. (A) Schematic illustration of human pancreatic ductal adenocarcinoma (PDAC) and pancreas tissue preparation, incubation with collagen-specific ⁶⁸Ga-CBP8 and control ⁶⁸Ga-CNBP probes, and autoradiography. (B) Representative autoradiography images of fresh resected human tissues and (C) heatmap of the autoradiography signal. (D-E) Quantitative comparison of autoradiography signal in the treated and untreated PDAC tissues and the patients' matched adjacent uninvolved pancreas (P) for ⁶⁸Ga-CBP8 (D) and ⁶⁸Ga-CNBP (E) (n = 8/group, unpaired t-test, two-tailed). (F) Representative H&E, Picro-Sirius Red (PSR), and type I collagen immunohistochemical staining of human tissues (scale bar: 200 µm). (G) Quantitative comparison of collagen proportional area (CPA) from PSR stained slides of human tumor and pancreas tissues (n = 8/group, unpaired t-test, two-tailed). (H) Correlation of the ⁶⁸Ga-CBP8 or ⁶⁸Ga-CNBP autoradiography signal with CPA. Each data point represents one tissue. RT: room temperature, a.u: arbitrary unit.

Figure 6

First-in-human evaluation of type I collagen PET in patients with PDAC. (A) Representative axial images of standard-of-care iodinated contrast-enhanced computed tomography (CT) on late arterial phase, gadolinium-enhanced magnetic resonance image (MRI) on portal venous phase, ⁶⁸Ga-CBP8 PET at 30-45 min post-injection, and fused PET and T1-weighted MR images in the axial plane are shown for a subject before and after standard chemoradiotherapy (CRT). PDAC tumor is shown with a white arrow. (B) Evaluation of dynamic PET from 0-90 min after injection of the ⁶⁸Ga-CBP8 shows rapid clearance of the probe from the blood (dashed red line), with blood SUV_mean reaching that of the uninvolved pancreas SUV_mean (blue line) at 15-30 min post-injection. Uptake to untreated PDAC is shown with black and to treated PDAC with dark red line. Data is shown in mean ± SEM. (C) The tumor SUV_mean is significantly higher than the pancreas at both pre-CRT and post-CRT scans (paired t-test). (D) PET uptake values at 30-60 min post-injection for the five patients who underwent PET scans at both time points show increased tumor SUV_mean in response to neoadjuvant CRT paired t-test). (E) Representative H&E and Picro-Sirius Red staining of the diagnostic core of tumor (T), and resected treated tumor and adjacent pancreas (P) tissues. (F) Quantitative analyses of histology show higher CPA in the treated compared to untreated tumors (** P = 0.0005 for all available tissues using unpaired t-test; P = 0.005 for 4 patients with available pre- and post-CRT tissues using paired t-test). (G) Significant positive correlation between the tissue collagen on histology (CPA) and SUV_mean.