Novel method for highly multiplexed gene expression profiling of circulating tumor cells (CTCs) captured from the blood of women with metastatic breast cancer
Abstract Background Enumeration of circulating tumor cells (CTCs) has proven clinical significance for monitoring patients with metastatic cancers. Multiplexed gene expression profiling of CTCs is a potential tool for assessing disease status and monitoring treatment response. The Parsortix® technol...
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2023-06-01
|
| Series: | Journal of Translational Medicine |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12967-023-04242-z |
| _version_ | 1797789539198042112 |
|---|---|
| author | Morvarid Farhang Ghahremani Kelly Kai Yin Seto Woohyun Cho Michael Craig Miller Paul Smith David Frederick Englert |
| author_facet | Morvarid Farhang Ghahremani Kelly Kai Yin Seto Woohyun Cho Michael Craig Miller Paul Smith David Frederick Englert |
| author_sort | Morvarid Farhang Ghahremani |
| collection | DOAJ |
| description | Abstract Background Enumeration of circulating tumor cells (CTCs) has proven clinical significance for monitoring patients with metastatic cancers. Multiplexed gene expression profiling of CTCs is a potential tool for assessing disease status and monitoring treatment response. The Parsortix® technology enables the capture and harvest of CTCs from blood based on cell size and deformability. The HyCEAD™ (Hybrid Capture Enrichment Amplification and Detection) assay enables simultaneous amplification of short amplicons for up to 100 mRNA targets, and the Ziplex™ instrument quantifies the amplicons for highly sensitive gene expression profiling down to single cell levels. The aim of the study was to functionally assess this system. Methods The HyCEAD/Ziplex platform was used to quantify the expression levels for 72 genes using as little as 20 pg of total RNA or a single cultured tumor cell. Assay performance was evaluated using cells or total RNA spiked into Parsortix harvests of healthy donor blood. The assay was also evaluated using total RNA obtained from Parsortix harvests of blood from metastatic breast cancer (MBC) patients or healthy volunteers (HVs). Results Using genes with low expression in WBC RNA and/or in unspiked Parsortix harvests from HVs, the assay distinguished between the different breast cancer and ovarian cancer cell lines with as little as 20 pg of total RNA (equivalent to a single cell) in the presence of 1 ng of WBC RNA. Single cultured cells spiked into Parsortix harvests from 10 mL of HV blood were also detected and distinguished from each other. CVs from repeatability experiments were less than 20%. Hierarchical clustering of clinical samples differentiated most MBC patients from HVs. Conclusion HyCEAD/Ziplex provided sensitive quantification of expression of 72 genes from 20 pg of total RNA from cultured tumor cell lines or from single cultured tumor cells spiked into lysates from Parsortix harvests of HV blood. The HyCEAD/Ziplex platform enables the quantification of selected genes in the presence of residual nucleated blood cells in Parsortix harvests. The HyCEAD/Ziplex platform is an effective tool for multiplexed molecular characterization of mRNA in small numbers of tumor cells harvested from blood. |
| first_indexed | 2024-03-13T01:52:10Z |
| format | Article |
| id | doaj.art-10e340c59b4e435cbb66c18214d09515 |
| institution | Directory Open Access Journal |
| issn | 1479-5876 |
| language | English |
| last_indexed | 2024-03-13T01:52:10Z |
| publishDate | 2023-06-01 |
| publisher | BMC |
| record_format | Article |
| series | Journal of Translational Medicine |
| spelling | doaj.art-10e340c59b4e435cbb66c18214d095152023-07-02T11:25:41ZengBMCJournal of Translational Medicine1479-58762023-06-0121111710.1186/s12967-023-04242-zNovel method for highly multiplexed gene expression profiling of circulating tumor cells (CTCs) captured from the blood of women with metastatic breast cancerMorvarid Farhang Ghahremani0Kelly Kai Yin Seto1Woohyun Cho2Michael Craig Miller3Paul Smith4David Frederick Englert5ANGLE Biosciences Inc.ANGLE Biosciences Inc.ANGLE Biosciences Inc.Clinical Development, ANGLE North America, Inc.ANGLE Biosciences Inc.ANGLE Biosciences Inc.Abstract Background Enumeration of circulating tumor cells (CTCs) has proven clinical significance for monitoring patients with metastatic cancers. Multiplexed gene expression profiling of CTCs is a potential tool for assessing disease status and monitoring treatment response. The Parsortix® technology enables the capture and harvest of CTCs from blood based on cell size and deformability. The HyCEAD™ (Hybrid Capture Enrichment Amplification and Detection) assay enables simultaneous amplification of short amplicons for up to 100 mRNA targets, and the Ziplex™ instrument quantifies the amplicons for highly sensitive gene expression profiling down to single cell levels. The aim of the study was to functionally assess this system. Methods The HyCEAD/Ziplex platform was used to quantify the expression levels for 72 genes using as little as 20 pg of total RNA or a single cultured tumor cell. Assay performance was evaluated using cells or total RNA spiked into Parsortix harvests of healthy donor blood. The assay was also evaluated using total RNA obtained from Parsortix harvests of blood from metastatic breast cancer (MBC) patients or healthy volunteers (HVs). Results Using genes with low expression in WBC RNA and/or in unspiked Parsortix harvests from HVs, the assay distinguished between the different breast cancer and ovarian cancer cell lines with as little as 20 pg of total RNA (equivalent to a single cell) in the presence of 1 ng of WBC RNA. Single cultured cells spiked into Parsortix harvests from 10 mL of HV blood were also detected and distinguished from each other. CVs from repeatability experiments were less than 20%. Hierarchical clustering of clinical samples differentiated most MBC patients from HVs. Conclusion HyCEAD/Ziplex provided sensitive quantification of expression of 72 genes from 20 pg of total RNA from cultured tumor cell lines or from single cultured tumor cells spiked into lysates from Parsortix harvests of HV blood. The HyCEAD/Ziplex platform enables the quantification of selected genes in the presence of residual nucleated blood cells in Parsortix harvests. The HyCEAD/Ziplex platform is an effective tool for multiplexed molecular characterization of mRNA in small numbers of tumor cells harvested from blood.https://doi.org/10.1186/s12967-023-04242-zParsortix®HyCEAD™Ziplex™CTCsBreast cancerMBC |
| spellingShingle | Morvarid Farhang Ghahremani Kelly Kai Yin Seto Woohyun Cho Michael Craig Miller Paul Smith David Frederick Englert Novel method for highly multiplexed gene expression profiling of circulating tumor cells (CTCs) captured from the blood of women with metastatic breast cancer Journal of Translational Medicine Parsortix® HyCEAD™ Ziplex™ CTCs Breast cancer MBC |
| title | Novel method for highly multiplexed gene expression profiling of circulating tumor cells (CTCs) captured from the blood of women with metastatic breast cancer |
| title_full | Novel method for highly multiplexed gene expression profiling of circulating tumor cells (CTCs) captured from the blood of women with metastatic breast cancer |
| title_fullStr | Novel method for highly multiplexed gene expression profiling of circulating tumor cells (CTCs) captured from the blood of women with metastatic breast cancer |
| title_full_unstemmed | Novel method for highly multiplexed gene expression profiling of circulating tumor cells (CTCs) captured from the blood of women with metastatic breast cancer |
| title_short | Novel method for highly multiplexed gene expression profiling of circulating tumor cells (CTCs) captured from the blood of women with metastatic breast cancer |
| title_sort | novel method for highly multiplexed gene expression profiling of circulating tumor cells ctcs captured from the blood of women with metastatic breast cancer |
| topic | Parsortix® HyCEAD™ Ziplex™ CTCs Breast cancer MBC |
| url | https://doi.org/10.1186/s12967-023-04242-z |
| work_keys_str_mv | AT morvaridfarhangghahremani novelmethodforhighlymultiplexedgeneexpressionprofilingofcirculatingtumorcellsctcscapturedfromthebloodofwomenwithmetastaticbreastcancer AT kellykaiyinseto novelmethodforhighlymultiplexedgeneexpressionprofilingofcirculatingtumorcellsctcscapturedfromthebloodofwomenwithmetastaticbreastcancer AT woohyuncho novelmethodforhighlymultiplexedgeneexpressionprofilingofcirculatingtumorcellsctcscapturedfromthebloodofwomenwithmetastaticbreastcancer AT michaelcraigmiller novelmethodforhighlymultiplexedgeneexpressionprofilingofcirculatingtumorcellsctcscapturedfromthebloodofwomenwithmetastaticbreastcancer AT paulsmith novelmethodforhighlymultiplexedgeneexpressionprofilingofcirculatingtumorcellsctcscapturedfromthebloodofwomenwithmetastaticbreastcancer AT davidfrederickenglert novelmethodforhighlymultiplexedgeneexpressionprofilingofcirculatingtumorcellsctcscapturedfromthebloodofwomenwithmetastaticbreastcancer |