Ex Vivo and In Vivo Imaging and Biodistribution of Aptamers Targeting the Human Matrix MetalloProtease-9 in Melanomas

International audienceThe human Matrix MetalloProtease-9 (hMMP-9) is overexpressed in tumors where it promotes the release of cancer cells thus contributing to tumor metastasis. We raised aptamers against hMMP-9, which constitutes a validated marker of malignant tumors, in order to design probes for imaging tumors in human beings. A chemically modified RNA aptamer (F3B), fully resistant to nucleases was previously described. This compound was subsequently used for the preparation of F3B-Cy5, F3B-S-acetylmercaptoacetyltriglycine (MAG) and F3B-DOTA. The binding properties of these derivatives were determined by surface plasmon resonance and electrophoretic mobility shift assay. Optical fluorescence imaging confirmed the binding to hMMP-9 in A375 melanoma bearing mice. Quantitative biodistribution studies were performed at 30 min, 1h and 2 h post injection of 99mTc-MAG-aptamer and 111In-DOTA-F3B. 99mTc radiolabeled aptamer specifically detected hMMP-9 in A375 melanoma tumors but accumulation in digestive tract was very high. Following i.v. injection of 111In-DOTA-F3B, high level of radioactivity was observed in kidneys and bladder but digestive tract uptake was very limited. Tumor uptake was significantly (student t test, p<0.05) higher for 111In-DOTA-F3B with 2.0%ID/g than for the 111In-DOTA-control oligonucleotide (0.7%ID/g) with tumor to muscle ratio of 4.0. Such difference in tumor accumulation has been confirmed by ex vivo scintigraphic images performed at 1h post injection and by autoradiography, which revealed the overexpression of hMMP-9 in sections of human melanomas. These results demonstrate that F3B aptamer is of interest for detecting hMMP-9 in melanoma tumor

Ex Vivo and In Vivo Imaging and Biodistribution of Aptamers Targeting the Human Matrix MetalloProtease-9 in Melanomas.

The human Matrix MetalloProtease-9 (hMMP-9) is overexpressed in tumors where it promotes the release of cancer cells thus contributing to tumor metastasis. We raised aptamers against hMMP-9, which constitutes a validated marker of malignant tumors, in order to design probes for imaging tumors in human beings. A chemically modified RNA aptamer (F3B), fully resistant to nucleases was previously described. This compound was subsequently used for the preparation of F3B-Cy5, F3B-S-acetylmercaptoacetyltriglycine (MAG) and F3B-DOTA. The binding properties of these derivatives were determined by surface plasmon resonance and electrophoretic mobility shift assay. Optical fluorescence imaging confirmed the binding to hMMP-9 in A375 melanoma bearing mice. Quantitative biodistribution studies were performed at 30 min, 1h and 2 h post injection of 99mTc-MAG-aptamer and 111In-DOTA-F3B. 99mTc radiolabeled aptamer specifically detected hMMP-9 in A375 melanoma tumors but accumulation in digestive tract was very high. Following i.v. injection of 111In-DOTA-F3B, high level of radioactivity was observed in kidneys and bladder but digestive tract uptake was very limited. Tumor uptake was significantly (student t test, p<0.05) higher for 111In-DOTA-F3B with 2.0%ID/g than for the 111In-DOTA-control oligonucleotide (0.7%ID/g) with tumor to muscle ratio of 4.0. Such difference in tumor accumulation has been confirmed by ex vivo scintigraphic images performed at 1h post injection and by autoradiography, which revealed the overexpression of hMMP-9 in sections of human melanomas. These results demonstrate that F3B aptamer is of interest for detecting hMMP-9 in melanoma tumor

Comparison of the results obtained by radiolabeling of representative tumor tissue sections with ¹¹¹In-DOTA-F3B aptamer (left image) and ¹¹¹In-DOTA-control sequence (right image).

<p>The difference of activity seems to increase in a tumor grade-dependent manner. (A) Lentigo malignant melanoma, (B) Nodular melanoma, (C) Mostly metastatic node.</p

<i>Ex Vivo</i> and <i>In Vivo</i> Imaging and Biodistribution of Aptamers Targeting the Human Matrix MetalloProtease-9 in Melanomas - Fig 4

<p>(A) Ventral planar fluorescence reflectance images acquired at 1h post injection of Cy5-F3B-aptamer (1, 2) or Cy5-control-sequence (3) into mice bearing human melanoma tumors. (B) Fluorescence reflectance imaging of organs after dissection of mice 2.</p

Quantitative biodistribution of ^99mTc-MAG-aptamer and ^99mTc-MAG-control aptamer as function of post i.v. injection delay expressed as % of injected dose per gram of tissue.

<p>Quantitative biodistribution of ^99mTc-MAG-aptamer and ^99mTc-MAG-control aptamer as function of post i.v. injection delay expressed as % of injected dose per gram of tissue.</p

Quantitative biodistribution of ¹¹¹In-DOTA-F3B-aptamer and ¹¹¹In-DOTA-control-aptamer as function of post i.v. injection delay expressed as % of injected dose per gram of tissue.

<p>Quantitative biodistribution of ¹¹¹In-DOTA-F3B-aptamer and ¹¹¹In-DOTA-control-aptamer as function of post i.v. injection delay expressed as % of injected dose per gram of tissue.</p

Immunostaining using anti-hMMP-9 murine monoclonal antibody.

<p>The antibody ab58803 localizing area of high expression of the integrin in respectively (A) Superficial Spread Melanoma, (B) Metastatic nodes, (C) Lentigo Malignant Melanoma. (D) Background in the immunohistochemical analysis of Superficial Spread Melanoma with staining of epidermis, endothelial cells and sebaceous glands. Immunohistochemical detection of hMMP-9 using ab58803 antibody in a mostly metastatic node, with specific cytoplasmic immunoreactivity in tumor cells (E) and negative results for conjunctive tissue and normal lymphocytes (F).</p

Ex vivo scintigraphic planar image of posterior paw at 1h after i.v. injection of ¹¹¹In-DOTA-F3B-aptamer and 111In-DOTA-control-aptamer.

<p>Ex vivo scintigraphic planar image of posterior paw at 1h after i.v. injection of ¹¹¹In-DOTA-F3B-aptamer and 111In-DOTA-control-aptamer.</p