Isolating human C cells by FACS for downstream -omics analysis to better understand the development of MTC in MEN2 patients

Jessica Fear; Guillaume E. Desanti; Katie Brookes; Naomi Richardson; Caitlin E.M. Thornton; Ling Zha; Vicki E. Smith; Martin L. Read; Renuka P. Dias; Christopher J. McCabe

doi:10.1186/s13044-024-00203-w

Abstract

Background: Multiple endocrine neoplasia type 2 (MEN2) is an inherited condition caused by RET proto-oncogene mutations and is responsible for approximately 20% of medullary thyroid cancer (MTC) cases. Predicting the onset of MTC in MEN2 patients is difficult and can vary between individuals in the same family with the same RET mutation. Enhanced molecular understanding of C cells via multi-omics approaches will be key to addressing the heterogeneity of RET mutational impacts. Previously, rat C cells have been isolated using Fluorescence-activated cell sorting (FACS), but this has not been reported for human C cells.

Aims: To establish a technique for isolating C-cells from fresh human thyroid tissues for -omics analysis to determine the individual risk factors influencing MTC development in MEN2 patients.

Methods: FACS was performed on fresh human primary thyroid tissues and mixed thyroid cell line populations to sort C cells, based on staining for the intracellular markers calcitonin and chromogranin A. TaqMan RT-PCR was used to measure the expression of calcitonin in FACS sorted cells to appraise their identity.

Results: FACS validations were initially performed on (i) two adult human primary thyroid samples, and (ii) mixed thyroid cell line populations containing 5% MTC-derived TT cells and 95% 8505C and TPC1 cells. Calcitonin, chromogranin A and double positive populations were isolated using FACS. Having established appropriate experimental conditions, FACS was subsequently performed on a primary human paediatric thyroid tissue sample which was successfully sorted into calcitonin-positive (24,214 cells), chromogranin A-positive (2,796 cells), double-positive (1,208 cells) and autofluorescent (21,860 cells) populations. TaqMan RT-PCR confirmed the expression of calcitonin in the sorted cell subgroups.

Conclusions: We designed a FACS protocol for isolating C cells for -omics analysis with future potential applications in understanding and improving personalised risk stratification in MEN2.

Original language	English
Article number	PO1
Number of pages	1
Journal	Thyroid Research
Volume	17
Issue number	S1
DOIs	https://doi.org/10.1186/s13044-024-00203-w
Publication status	Published - 14 Jun 2024
Event	72nd Annual Meeting of the British Thyroid Association - Royal College of Pathologists, London, United Kingdom Duration: 14 Jun 2024 → 14 Jun 2024 https://www.british-thyroid-association.org/sandbox/bta2016/june_6_2024bta_annualmeeting-programme__final.pdf

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SDG 3 Good Health and Well-being

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10.1186/s13044-024-00203-wLicence: Creative Commons: Attribution (CC BY)

1 Finished
2 Active

MRC IAA: A new strategy to enhance the radioiodine treatment of thyroid cancer
McCabe, C. (Principal Investigator)
Medical Research Council
1/03/24 → 30/09/26
Project: Research Councils
Making Radioiodine Treatment a Realistic Therapeutic Opportunity in Breast Cancer
McCabe, C. (Principal Investigator)
US ARMY
30/09/21 → 29/09/26
Project: Research
Wellcome TDF: Utilising an -omics approach for personalised risk stratification in MEN2
Dias, R. (Principal Investigator), Boelaert, K. (Co-Investigator) & McCabe, C. (Co-Investigator)
The Wellcome Trust
1/04/23 → 31/03/24
Project: Research

Cite this

@article{077a8ca000c6491bba497110f16a5c84,

title = "Isolating human C cells by FACS for downstream -omics analysis to better understand the development of MTC in MEN2 patients",

abstract = "Background: Multiple endocrine neoplasia type 2 (MEN2) is an inherited condition caused by RET proto-oncogene mutations and is responsible for approximately 20\% of medullary thyroid cancer (MTC) cases. Predicting the onset of MTC in MEN2 patients is difficult and can vary between individuals in the same family with the same RET mutation. Enhanced molecular understanding of C cells via multi-omics approaches will be key to addressing the heterogeneity of RET mutational impacts. Previously, rat C cells have been isolated using Fluorescence-activated cell sorting (FACS), but this has not been reported for human C cells. Aims: To establish a technique for isolating C-cells from fresh human thyroid tissues for -omics analysis to determine the individual risk factors influencing MTC development in MEN2 patients. Methods: FACS was performed on fresh human primary thyroid tissues and mixed thyroid cell line populations to sort C cells, based on staining for the intracellular markers calcitonin and chromogranin A. TaqMan RT-PCR was used to measure the expression of calcitonin in FACS sorted cells to appraise their identity. Results: FACS validations were initially performed on (i) two adult human primary thyroid samples, and (ii) mixed thyroid cell line populations containing 5\% MTC-derived TT cells and 95\% 8505C and TPC1 cells. Calcitonin, chromogranin A and double positive populations were isolated using FACS. Having established appropriate experimental conditions, FACS was subsequently performed on a primary human paediatric thyroid tissue sample which was successfully sorted into calcitonin-positive (24,214 cells), chromogranin A-positive (2,796 cells), double-positive (1,208 cells) and autofluorescent (21,860 cells) populations. TaqMan RT-PCR confirmed the expression of calcitonin in the sorted cell subgroups. Conclusions: We designed a FACS protocol for isolating C cells for -omics analysis with future potential applications in understanding and improving personalised risk stratification in MEN2.",

author = "Jessica Fear and Desanti, \{Guillaume E.\} and Katie Brookes and Naomi Richardson and Thornton, \{Caitlin E.M.\} and Ling Zha and Smith, \{Vicki E.\} and Read, \{Martin L.\} and Dias, \{Renuka P.\} and McCabe, \{Christopher J.\}",

year = "2024",

month = jun,

day = "14",

doi = "10.1186/s13044-024-00203-w",

language = "English",

volume = "17",

journal = "Thyroid Research",

issn = "1756-6614",

publisher = "BioMed Central",

number = "S1",

note = "72nd Annual Meeting of the British Thyroid Association ; Conference date: 14-06-2024 Through 14-06-2024",

url = "https://www.british-thyroid-association.org/sandbox/bta2016/june\_6\_2024bta\_annualmeeting-programme\_\_final.pdf",

}

TY - JOUR

T1 - Isolating human C cells by FACS for downstream -omics analysis to better understand the development of MTC in MEN2 patients

AU - Fear, Jessica

AU - Desanti, Guillaume E.

AU - Brookes, Katie

AU - Richardson, Naomi

AU - Thornton, Caitlin E.M.

AU - Zha, Ling

AU - Smith, Vicki E.

AU - Read, Martin L.

AU - Dias, Renuka P.

AU - McCabe, Christopher J.

PY - 2024/6/14

Y1 - 2024/6/14

N2 - Background: Multiple endocrine neoplasia type 2 (MEN2) is an inherited condition caused by RET proto-oncogene mutations and is responsible for approximately 20% of medullary thyroid cancer (MTC) cases. Predicting the onset of MTC in MEN2 patients is difficult and can vary between individuals in the same family with the same RET mutation. Enhanced molecular understanding of C cells via multi-omics approaches will be key to addressing the heterogeneity of RET mutational impacts. Previously, rat C cells have been isolated using Fluorescence-activated cell sorting (FACS), but this has not been reported for human C cells. Aims: To establish a technique for isolating C-cells from fresh human thyroid tissues for -omics analysis to determine the individual risk factors influencing MTC development in MEN2 patients. Methods: FACS was performed on fresh human primary thyroid tissues and mixed thyroid cell line populations to sort C cells, based on staining for the intracellular markers calcitonin and chromogranin A. TaqMan RT-PCR was used to measure the expression of calcitonin in FACS sorted cells to appraise their identity. Results: FACS validations were initially performed on (i) two adult human primary thyroid samples, and (ii) mixed thyroid cell line populations containing 5% MTC-derived TT cells and 95% 8505C and TPC1 cells. Calcitonin, chromogranin A and double positive populations were isolated using FACS. Having established appropriate experimental conditions, FACS was subsequently performed on a primary human paediatric thyroid tissue sample which was successfully sorted into calcitonin-positive (24,214 cells), chromogranin A-positive (2,796 cells), double-positive (1,208 cells) and autofluorescent (21,860 cells) populations. TaqMan RT-PCR confirmed the expression of calcitonin in the sorted cell subgroups. Conclusions: We designed a FACS protocol for isolating C cells for -omics analysis with future potential applications in understanding and improving personalised risk stratification in MEN2.

AB - Background: Multiple endocrine neoplasia type 2 (MEN2) is an inherited condition caused by RET proto-oncogene mutations and is responsible for approximately 20% of medullary thyroid cancer (MTC) cases. Predicting the onset of MTC in MEN2 patients is difficult and can vary between individuals in the same family with the same RET mutation. Enhanced molecular understanding of C cells via multi-omics approaches will be key to addressing the heterogeneity of RET mutational impacts. Previously, rat C cells have been isolated using Fluorescence-activated cell sorting (FACS), but this has not been reported for human C cells. Aims: To establish a technique for isolating C-cells from fresh human thyroid tissues for -omics analysis to determine the individual risk factors influencing MTC development in MEN2 patients. Methods: FACS was performed on fresh human primary thyroid tissues and mixed thyroid cell line populations to sort C cells, based on staining for the intracellular markers calcitonin and chromogranin A. TaqMan RT-PCR was used to measure the expression of calcitonin in FACS sorted cells to appraise their identity. Results: FACS validations were initially performed on (i) two adult human primary thyroid samples, and (ii) mixed thyroid cell line populations containing 5% MTC-derived TT cells and 95% 8505C and TPC1 cells. Calcitonin, chromogranin A and double positive populations were isolated using FACS. Having established appropriate experimental conditions, FACS was subsequently performed on a primary human paediatric thyroid tissue sample which was successfully sorted into calcitonin-positive (24,214 cells), chromogranin A-positive (2,796 cells), double-positive (1,208 cells) and autofluorescent (21,860 cells) populations. TaqMan RT-PCR confirmed the expression of calcitonin in the sorted cell subgroups. Conclusions: We designed a FACS protocol for isolating C cells for -omics analysis with future potential applications in understanding and improving personalised risk stratification in MEN2.

U2 - 10.1186/s13044-024-00203-w

DO - 10.1186/s13044-024-00203-w

M3 - Abstract

SN - 1756-6614

VL - 17

JO - Thyroid Research

JF - Thyroid Research

IS - S1

M1 - PO1

T2 - 72nd Annual Meeting of the British Thyroid Association

Y2 - 14 June 2024 through 14 June 2024

ER -

Isolating human C cells by FACS for downstream -omics analysis to better understand the development of MTC in MEN2 patients

Abstract

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MRC IAA: A new strategy to enhance the radioiodine treatment of thyroid cancer

Making Radioiodine Treatment a Realistic Therapeutic Opportunity in Breast Cancer

Wellcome TDF: Utilising an -omics approach for personalised risk stratification in MEN2

Cite this