A novel HER2 protein identification methodology in breast cancer cells using Raman spectroscopy and Raman imaging: an analytical validation study

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Abstract

Background

Conventional assays such as immunohistochemistry (IHC) and in situ hybridization (ISH) used in clinical procedures for quantification of the human epidermal growth factor receptor-2 (HER2) status in breast cancer have many limitations. Our results suggest that a new Raman method may improve specificity that will result in better patients selection for HER2 targeted therapy. In the current study, we have used HER2 expression in a broad range of breast cancer phenotypes to explore the potential utility of a novel immunodetection technique, using Raman spectroscopy and Raman imaging combined with artificial intelligence models.

Methods

The expression of HER2 protein in different cancer subtypes was evaluated using Raman methodology to test correlations with currently used quantitative protein analysis for a broad range of cancer subtypes based on the expression levels of estrogen and progesterone receptors (ER and PR), HER2, cytokeratins and claudins. In the current study, five breast cancer cell lines: MCF-10A, MCF-7, MDA-MB-231, HTB-30 (SK-BR-3) and AU-565 were tested.

Results

The correlations between Raman method and conventional HER2 testing methodologies (IHC and ISH) have been tested. Raman measurements showed a strong linear correlation ( p = 0.05, R 2 =0,9816) with IHC analysis in the studied breast cell lines: MCF-10A, MCF-7, MDA-MB- 231, HTB-30 (SK-BR-3) and AU-565 representing normal, non-tumorigenic epithelial cells, triple-positive breast carcinoma and triple-negative breast cancer cell lines.

Conclusions

Analytic testing of Raman spectroscopy and Raman imaging demonstrated that this method may offer advantages over currently used diagnostic methodologies. The broad range of HER2 expression on the surface of human breast normal and cancer cells from triple negative to triple positive cell lines were studied. Our data demonstrate that Raman based methods for HER2 quantitation of HER2 may offer significant progress in patient selection for HER2 targeted therapy over conventional HER2 identification. Further studies of HER2 determination by Raman methods with in vivo cells and ex vivo tissue are warranted.

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  1. Abstract

    I enjoyed this paper! I'm excited to try detecting tyrosine phosphorylation using Raman in my own work. I have a couple of general suggestions: one is to use color (or add clearer keys) in the figures to differentiate between HER2+ and HER2- cell types, so that it is easier to understand the results. I also think this paper could use another round of general copyediting - Grammarly offers a free version of their software that can make this step very fast.

  2. No ligands for HER2 have yet been identified yet. 21 22 and dimerization with any of the other three subdomains is considered to activate HER2. 23 The dimerization in the extracellular region of HER2 induces intracellular conformational changes that trigger tyrosine kinase activation.

    This sentence is included verbatim in the introduction.

  3. Fig. 9 shows average normalized Raman intensity at 1618 cm-1 at membranes (A,B) and mitochondria (B) in breast cancer cells: triple-positive MCF-7 (B), (HTB-30) and AU-565 (C) overexpressing HER2, the normal cells (MCF-10A) (HER2 at the normal level) and triple - negative aggressive breast cancer (MDA-MB-231)

    I'm not sure what the letters in parentheses refer to here.

  4. One can see that that the highest concentration of cytochrome c represented by the vibration at 1584 cm-1 is located in mitochondria and support the results reported recently1

    The link for this citation is missing, but I would like to read it!

  5. Raman spectra of a typical breast cancer cell (HTB-30) for the cell organelles (nucleus (red), endoplasmic reticulum (blue), lipid droplets (orange), cytoplasm (green), mitochondria (magenta), membrane (light grey).

    For Figs 5 and 6, it would be helpful to include descriptions of each panel in the description & reference them directly in the text.

  6. The presented in Fig. 7 cells have HER2 protein expression on the surface of the cell that belongs to a family of receptor tyrosine kinases (RTKs) and consists of an extracellular domain that includes four subdomains (I-IV) [16], a single helix transmembrane lipophilic segment and an intracellular region that contains a tyrosine kinase domain (TKD).

    I'm not sure why this is mentioned here, it is established in the introduction. Unless this is implying that you can acquire all of this info directly from the Raman spectra?

  7. Fig. 5C shows the Raman bands of cytochrome c at 1582 cm-1 and amide I at 1656 cm-1 of a typical breast cancer cell (HTB-30).

    I believe this sentence is actually referencing Fig 5B.

  8. As it is well known these processes regulate efficiency of the oxidative phosphorylation and is directly related to many human diseases, including cancer, through a lack of energy, ROS production, cytochrome c release, and activation of apoptosis.

    I think this sentence may also have a typo - there is no follow up phrase to "As it is well known that X..." What did this knowledge lead you to?

  9. It has been proposed that reversible phosphorylation of cytochrome c mediated by cell signaling pathways is primary regulatory mechanism in living species that determines mitochondrial respiration, electron transport chain (ETC) flux, proton gradient ΔΨm, ATP production, and ROS generation

    Proposed by who? I think this claim could use a citation so that readers can look further into the evidence supporting it.

  10. Our data demonstrate that Raman based methods for HER2 quantitation of HER2 may offer significant progress in patient selection for HER2 targeted therapy over conventional HER2 identification.

    I think this sentence may have a typo - it says "HER2 quantitation of HER2," rather than just "quantitation of HER2" or "HER2 quantitation."