The aim of the study was to analyze the specificity and sensitivity of highly fluorescent quantum dots (QDs) conjugated to tumour specific single domain antibodies, one directed against the human epidermal growth factor receptor 2 (HER2-QDs) and the other directed against the human carcinoembryonic antigen (CEA-QDs) to detect disseminated HER2 and CEA positive tumour cells in ex vivo biological samples of metastatic tumour mouse models. The following metastatic breast tumour models were used: 1) HER2 positive human BT-474 cells were orthotopically implanted into nu/nu mice that had received subcutaneous hormone-pellets; 2) human HER2 positive SKBR3 cells were implanted into the liver of neonatal immunodeficientNOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice (JAX®) that were irradiated with 1 Gy for 3 hours before transplantation. HER2 negative breast tumours that developed after orthotopic implantation of MDA-MB231cells were used as controls. Orthotopic pancreatic tumour models consisted of nu/nu mice that received either CEA positive human AsPC1 or Capan1 tumour cells or CEA negative human MiaPaca cells as negative controls. After tumour growth, biological samples were excised, fixed overnight in formalin, and embedded in paraffin or in 5% agarose. Staining was performed either with the HER2-QDs and CEA-QDs or with commercially available anti HER2 or anti CEA antibodies. Images of the immunofluorescence stained sections were acquired with a Leica SP5 Confocal Laser Scanning Microscopy (Leica Germany) and a custom-made 2-Photon Laser Scanning Microscopy equipped with an fs-pulsed titanium-sapphire laser (Chameleon Ultra II, Coherent).
By immunostaining with HER2- and CEA-QDs of either 5-10 µm paraffin sections or 50 µm agarose sections we were able to detect HER2 and CEA positive disseminated human tumour cells in different mouse organs including brain, testis, lung, liver and lymph nodes. All controls, including the use of unconjugated QDs, QDs conjugated with protein GP120, staining of MDA-MB231 and MiaPacaxenografts with the probes were negative.
Our results demonstrate that the HER2 and CEA -QDs conjugates were highly specific and sensitive in detecting disseminated human tumour cells in mouse tissue. The advantages of using QDs over the commercial fluorophores include high intensity signal, reduced autofluorescence, better tissue penetration and less bleaching.
In conclusion, this nanoprobe based technology may become a powerful tool to detect disseminated tumour cells in biological samples and thereby help to diagnose early cancer or cancer progression by assessing the metastatic spread.