Laser Capture Proteomics: spatial tissue molecular profiling from the bench to personalized medicine

Abstract

Introduction: Laser Capture Microdissection (LCM) uses a laser to isolate, or capture, specific cells of interest in a complex heterogeneous tissue section, under direct microscopic visualization. Recently, there has been a surge of publications using LCM for tissue spatial molecular profiling relevant to a wide range of research topics.

Areas covered: We summarize the many advances in tissue Laser Capture Proteomics (LCP) using mass spectrometry for discovery, and protein arrays for signal pathway network mapping. This review emphasizes: a) transition of LCM phosphoproteomics from the lab to the clinic for individualized cancer therapy, and b) the emerging frontier of LCM single cell molecular analysis combining proteomics with genomic, and transcriptomic analysis. The search strategy was based on the combination of MeSH terms with expert refinement.

Expert opinion: LCM is complemented by a rich set of instruments, methodology protocols, and analytical A.I. (artificial intelligence) software for basic and translational research. Resolution is advancing to the tissue single cell level. A vision for the future evolution of LCM is presented. Emerging LCM technology is combining digital and AI guided remote imaging with automation, and telepathology, to a achieve multi-omic profiling that was not previously possible.

Keywords: DCIS (Ductal Carcinoma in Situ); ERBB (Epidermal growth factor receptor family); Laser Capture Microdissection; kinase signaling; mass spectrometry; neoadjuvant therapy; phosphoprotein; proteomics; tissue spatial profiling; tumor-host microenvironment.

Figure 1.

Spatially Resolved Tissue Proteomics Technology Categories. Example major classes of technologies that exist for spatially (in situ) resolved proteomics: A) Analyte probe labeled or label-free microscopic imaging of tissue: Example technologies that fall under this category include standard clinical immunohistochemistry using one antibody probe at a time, multiplex sequential or parallel antibody probes, and label free imaging. B) Surface scanning or grid-based surface elution of labeled or unlabeled tissue molecules. Example technologies that fall under this category include scanning MALDI, Tissue laser scanning after the tissue or cells are labeled with heavy metal tagged antibodies (CyTOF), or antibody-labeled nucleic acid barcoded tags (NanoString nCounter), or D) Isolation or capture of full thickness tissue regions of interest followed by external extraction and analysis. Example technologies that fall under this category include Laser Capture Microdissection LCM using ultraviolet laser cutting around the perimeter of the region, or cell of interest, or direct full thickness removal by infrared laser capture embedding onto a capture surface or both UV and IR laser methods combined together.

Figure 2.

LCM technology uses laser cutting or laser induced surface capture (upper insert) to procure select tissue full thickness sample. The procured material is then extracted and subjected to a plurality of downstream analytical tools yielding discovery of spatially localized tissue molecules or multiplex quantitation of known analytes.

Figure 3.

Phosphorylated epitopes indicative of ERBB2 (HER2) family signal activity are correlated with pathologic complete response (pCR). Dimerization (homotypic or heterotypic) following ligand engagement triggers autophosphorylation. The phosphorylated sites on the cytoplasmic domain dock with downstream pathways indicated in green and yellow. Receptor kinase function, and downstream signaling driving the cancer cell can be blocked by Pertuzumab, T-DM-1, or Trastuzumab, on the cell surface domain, or by kinase inhibitors Neratinib and Lapatinib that act at the level of the cytoplasmic domain(s). Unblinding of the RPPA data revealed that the most significant phosphorylated epitopes predictive of pCR were located in the distal cytoplasmic domain of the ERBB1 receptor (outlined in the red box) that a) is the known target of Neratinib, and b) are docking sites for triggering downstream growth and survival pathways driving the breast cancer cell. Insert: 2D scatterplot indicating clear separation of pCR yes(green)/no(red). from Wulkuhle et al [31]

Figure 4.

Depiction of how the proposed HER2 pathway activation test conducted on the diagnostic biopsy can be employed in the near future for personalized escalation and de-escalation therapies that have recently been clinically validated.

Figure 5.

Digital pathology vision of the future. A diagnostic biopsy pathologic tissue section histologic image is digitally captured at high resolution, sent to the cloud, and then marked up remotely on a scientist or clinician’s computer screen (left panel). Once the specific histopathology regions are marked for interrogation on-screen they are remotely and automatically microdissected (right panel) and analyzed. Improved liquid cover slip chemistry and high resolution UV capture permit capture and analysis of the same tissue cells that are imaged at the microscopic level. Data from the molecular analysis of each selected region is then ported back through the cloud and can be viewed on-screen (left panel) .