L.D e Souza, N.Y-W. Tang, R.O. do Nascimento, S. Mal, L. Gaboury, R. Martel
Université de Montréal - Institute for Research in Immunology and Cancer (IRIC),
Canada
Keywords: breast cancer, ER, HER2, SWCNT, Raman spectroscopy
Summary:
Breast cancer is the most common cancer in Canadian women. Currently available diagnosis is based on immunohistochemistry (IHC) to identify cells expressing estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). This technique targets cancer cells to determine the different molecular subtypes and requires one tissue section per marker analyzed. With the advent of immunotherapy, determination of infiltrated T lymphocytes in tumor microenvironment is a crucial step to guide the personalized treatment in combination with chemotherapy. Until now, there is no option to detect all these markers in the same tissue section. The detection all of them separated is not realistic in clinics and would delay the diagnosis, which impairs the application of these tumor landscapes as a routine. Hence, the development of simultaneous detection in one tissue section is more than necessary to achieve the safe implementation of personalized treatment. Raman spectroscopy imaging with Raman probes emerges as a method of choice for a simultaneous detection of these markers. Raman probes have a unique spectrum, peaks widths of about 2 nm and non-bleaching signal. They are based on single-walled carbon nanotubes (SWCNT) of ~200 nm length and 1.4 nm diameter. Each tube contains internalized fluorescent dye (β-carotene and sexithiophene) emitting a specific Raman signature (fig. 3) used to perform simultaneous detection. The SWCNT were functionalized with an amine-polyethylene glycol- carboxylic acid (NH2-PEG-COOH) to allow the attachment of antibodies anti-ER and anti-HER2 (fig.1) to detect these markers. Immunogold labeling confirmed the attachment of the antibody to SWCNT in the right orientation. These Raman probes were then used in formalin-fixed paraffin-embedded (FFPE) tissue samples of breast cancer selected from Histology’s platform biobank. ER+ and HER2+ samples were identified by IHC and selected for Raman spectroscopy. Cell lines MCF-7 (ER+) and SKBR-3 (HER2+) were also used as controls. IHC results using tissue and cell lines were compared to Raman imaging to evaluate probes sensitivity and specificity. Preliminary results with Raman probes show that anti-ER and anti-HER2 attach to the samples, in both tissue (fig 2) and cells (fig 3); however the Raman signal is weak, compared to the sensitivity of IHC. Currently we are optimizing the Raman probe to obtain a better signal, as well as optimizing the Raman probe incubation with tissues.