Chaperonin containing TCP1 as a marker for identification of circulating tumor cells in blood

Abstract

Herein we report the use of Chaperonin-Containing TCP-1 (CCT or TRiC) as a marker to detect circulating tumor cells (CTCs) that are shed from tumors during oncogenesis. Most detection methods used in liquid biopsy approaches for enumeration of CTCs from blood, employ epithelial markers like cytokeratin (CK). However, such markers provide little information on the potential of these shed tumor cells, which are normally short-lived, to seed metastatic sites. To identify a marker that could go beyond enumeration and provide actionable data on CTCs, we evaluated CCT. CCT is a protein-folding complex composed of eight subunits. Previously, we found that expression of the second subunit (CCT2 or CCTβ) inversely correlated with cancer patient survival and was essential for tumorigenesis in mice, driving tumor-promoting processes like proliferation and anchorage-independent growth. In this study, we examined CCT2 expression in cancer compared to normal tissues and found statistically significant increases in tumors. Because not all blood samples from cancer patients contain detectable CTCs, we used the approach of spiking a known number of cancer cells into blood from healthy donors to test a liquid biopsy approach using CCT2 to distinguish rare cancer cells from the large number of non-cancer cells in blood. Using a clinically validated method for capturing CTCs, we evaluated detection of intracellular CCT2 staining for visualization of breast cancer and small cell lung (SCLC) cancer cells. We demonstrated that CCT2 staining could be incorporated into a CTC capture and staining protocol, providing biologically relevant information to improve detection of cancer cells shed in blood. These results were confirmed with a pilot study of blood from SCLC patients. Our studies demonstrate that detection of CCT2 could identify rare cancer cells in blood and has application in liquid biopsy approaches to enhance the use of minimally invasive methods for cancer diagnosis.

Chaperonin containing TCP-1 (CCT/TRiC) is a novel therapeutic and diagnostic target for neuroblastoma

“…To directly evaluate CCT2 levels in tumor tissues, we used a pediatric cancer tissue microarray (TMA) for examining CCT2 protein by standard IHC. A clinical/anatomic pathologist on our team previously developed an IHC scoring scheme from 0-4 for CCT2 staining of tissues, with normal tissues staining usually in the 0-1 range and tumor tissues staining >1, based on the intensity of the cytoplasmic and nuclear stains ( 21 , 26 , 32 ). Results from the TMA revealed strong CCT2 staining (scores 3-4) across most of the tumor tissues, representative images are shown in Figure 2A , with the full TMA shown in Figure S2 .…”

“…CellSearch is an FDA-approved enrichment method for enumeration of CTCs in the prognosis and monitoring of breast, colon, and prostate cancer ( 43 , 44 ). Previously our lab modified the CellSearch CXC kit to incorporate the intracellular staining of CCT2 in the automated protocol that stains for cytokeratins 8, 18, and 19 (CKs), CD45, and DAPI ( 32 ). The CellSearch System uses ferrofluid or iron particles conjugated to an anti-epithelial cellular adhesion molecule (EpCAM) antibody to magnetically capture and initially enrich CTCs from blood.…”

“…Anti-CCTβ antibody [amino acids 277 and 473 of Human TCP1 beta] (LifeSpan Biosciences) was used. Slides were stained and scored for the CCT2 stain by an independent and identification-blinded pathologist, as described previously ( 21 , 32 ). Digital images were acquired at 40X magnification using the BZ-X800 Keyence.…”

“…The antibody for CCT2-phycoerythrin (PE): [NP_006422] (LSBio) was added at a concentration of 24 μg/mL. Cells were analyzed through the CellSearch Analyzer II for CTCs and CCT2-positive (CCT2+) cancer cells as described previously ( 32 ). Blood collection was conducted by observing the human subject protection criteria for the University of Central Florida (UCF) and was approved by the Institutional Review Board (IRB) committees at UCF.…”

“…The manual staining protocol for cancer cells in buffer for antibody optimization was adapted from Lowes et al (35). IMR-32 cells in 5% FBS/PBS were stained according to the protocol previously described (32), with the following modification: IMR-32 cells were washed twice with dilution buffer post-antibody staining. After staining, cells were re-suspended in dilution buffer and transferred into CellSearch MagNest cartridges and run in the CellSearch Analyzer II, and evaluated for CCT2positive cells as described previously (32).…”