Selective ablation of TRA-1-60+ pluripotent stem cells suppresses tumor growth of prostate cancer

Abstract

Purpose: TRA-1-60 (TRA) is an established transcription factor of embryonic signaling and a well-known marker of pluripotency. It has been implicated in tumorigenesis and metastases, is not expressed in differentiated cells, which makes it an appealing biomarker for immunopositron emission tomography (immunoPET) imaging and radiopharmaceutical therapy (RPT). Herein, we explored the clinical implications of TRA in prostate cancer (PCa), examined the potential of TRA-targeted PET to specifically image TRA⁺ cancer stem cells (CSCs) and assessed response to the selective ablation of PCa CSCs using TRA-targeted RPT. Experimental Design: First, we assessed the relationship between TRA (PODXL) copy number alterations (CNA) and survival using publicly available patient databases. The anti-TRA antibody, Bstrongomab, was radiolabeled with Zr-89 or Lu-177 for immunoPET imaging and RPT in PCa xenografts. Radiosensitive tissues were collected to assess radiotoxicity while excised tumors were examined for pathologic treatment response. Results: Patients with tumors having high PODXL CNA exhibited poorer progression-free survival than those with low PODXL, suggesting that it plays an important role in tumor aggressiveness. TRA-targeted immunoPET imaging specifically imaged CSCs in DU-145 xenografts. Tumors treated with TRA RPT exhibited delayed growth and decreased proliferative activity, marked by Ki-67 immunohistochemistry. Aside from minor weight loss in select animals, no significant signs of radiotoxicity were observed in the kidneys or livers. Conclusions: We successfully demonstrated the clinical significance of TRA expression in human PCa, engineered and tested radiotheranostic agents to image and treat TRA⁺ prostate CSCs. Ablation of TRA⁺ CSCs blunted PCa growth. Future studies combining CSC ablation with standard treatment will be explored to achieve durable responses.

Keywords: Lutetium-177; TRA-1-60; immunoPET imaging; prostate cancer stem cells; radionuclide therapy.

Figure 1

The Kaplan-Meier curve of A. progression-free survival (PFS) by copy number alterations (CNA) of PODXL (High vs. Low, Low as reference) of primary PCa patients respectively. CNA was dichotomized into two groups (High vs. Low) by the median for PFS. The p-value was obtained by a log-rank test. HR and CI stand for 'hazard ratio', and 'confidence interval', respectively. C. TRA⁺ cells (brown) are shown infiltrating lymph node tissue of a metastatic prostate cancer patient.

Figure 2

In vivo PET Imaging and ex vivo analysis. A. PET images obtained with [⁸⁹Zr]Zr-DFO-Bsg from 24 to 120 h p.i. demonstrates sustained tracer accumulation in DU-145 tumors (white circle, T=tumor, H=heart). B. Accumulation of [⁸⁹Zr]Zr-DFO-IgG is lower than the TRA-specific probe in a separate cohort of mice. C. Ex vivo autoradiography of DU-145 exhibits focal heterogeneous localization of the tracer. The white box represents area where tracer uptake was measured. D. Immunohistochemistry of tumor sections (60 μm, 40x) display more intense brown staining (black arrows) indicating TRA expression. E. A Spearman correlation analysis comparing tracer uptake in the tumor (nCi) vs. %TRA-1-60 staining by IHC showed a positive correlation of r = 0.78.

Figure 3

Co-expression of TRA-1-60 (TRA), CD133, and CD44 in DU145 tumor. Heterogeneous DU-145 tumor sections stained for either CD133 (green), CD44 (green) or TRA (red). DAPI (blue) was used to identifying nuclei. Representative cells both expressing CD44/TRA or CD133/TRA were marked with white arrowheads. Scale bar, 50 µm.

Figure 4

Tumorigenicity of TRA⁺ cells. A. TRA⁺ and TRA^- cells sorted from DU-145 tumors were re-implanted in mice. Tumors developed faster and at higher incidence with TRA⁺ enriched versus negative cells. B. Ex vivo analysis of %TRA⁺ cells from TRA⁺ sorted and heterogeneous xenografts. C. TRA⁺ xenografts imaged at 48 h p.i. of [⁸⁹Zr]Zr-DFO-Bsg displayed tumor (white circle) accumulation D. A Spearman correlation of tracer uptake versus tumor volume in TRA⁺ sorted xenografts shows a strong positive correlation.

Figure 5

Radiopharmaceutical therapy with [¹⁷⁷Lu]Lu-CHX-A”-DTPA-Bsg. A. SPECT/CT images display tumor accumulation with clearance from non-specific tissues after 120 h p.i. B. Tissue distribution at 120 h p.i. C. Tumor growth is suppressed in mice administered with high (27.8 MBq) and low (18.5 MBq) activities. D. Unmodified Bsg alone does not elicit therapeutic response.

Figure 6

TRA-1-60 (TRA) expression and proliferative activity of DU-145 tumors after treatment. A. TRA expression (red) is diminished after treatment with 18.5 MBq (middle) and 27.8 MBq (bottom) activities of [¹⁷⁷Lu]Lu-CHX-A”-DTPA-Bsg compared to control (top). Cell proliferation displayed via Ki-67 immunostaining (left panels) show decreased proliferative activity in treated tumors. B. Quantification of Ki67 marked decreased proliferation in both treated groups in a dose-dependent manner versus control untreated tumors. *P = 0.0425, **P = 0.0010, ****P<0.0001

Figure 7

Hematology (CBC) and serum chemistry analysis of DU-145 xenograft mice administered with high activity (27.8 MBq) [¹⁷⁷Lu]Lu-CHX-A”-DTPA-Bsg. A. WBC B. RBC C. platelets, D. hemoglobin (HGB), E. hematocrit (HCT), F. blood urea nitrogen (BUN), G. alanine aminotransferase (ALT), H. alkaline phosphatase (ALP). Assessment of % weight changes in I. control and treated mice at J. 18.5 MBq and K. 27.8 MBq.

Figure 8

Histologic evaluation via H&E. Representative renal tissue from A. control and B. treated mice (H&E, 20×). Representative liver tissue from C. control and D. treated mice (H&E, 10×).