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3D cultivation of non-small-cell lung cancer cell lines using four different methods

Malmros, Karina LU ; Kirova, Nadi LU ; Kotarsky, Heike LU ; Carlsén, Daniel ; Mansour, Mohammed S I LU orcid ; Magnusson, Mattias LU ; Prabhala, Pavan LU and Brunnström, Hans LU orcid (2024) In Journal of Cancer Research and Clinical Oncology 150. p.1-10
Abstract

PURPOSE: The aim of this study was to set up reliable and reproducible culture conditions for 3D tumoroids derived from non-small cell lung cancer (NSCLC) cell lines to enable greater opportunity for successful cultivation of patient-derived samples.

METHODS: Four NSCLC cell lines, two adenocarcinomas (A549, NCI-H1975) and two squamous cell carcinomas (HCC-95, HCC-1588), were first cultured in traditional 2D settings. Their expected expression profiles concerning TTF-1, CK7, CK5, and p40 status were confirmed by immunohistochemistry (IHC) before the generation of 3D cultures. Tumoroids were established in the hydrogel GrowDex ®-T, Nunclon™ Sphera™ flasks, BIOFLOAT™ plates, and Corning ® Elplasia ® plates. Western blot was used to... (More)

PURPOSE: The aim of this study was to set up reliable and reproducible culture conditions for 3D tumoroids derived from non-small cell lung cancer (NSCLC) cell lines to enable greater opportunity for successful cultivation of patient-derived samples.

METHODS: Four NSCLC cell lines, two adenocarcinomas (A549, NCI-H1975) and two squamous cell carcinomas (HCC-95, HCC-1588), were first cultured in traditional 2D settings. Their expected expression profiles concerning TTF-1, CK7, CK5, and p40 status were confirmed by immunohistochemistry (IHC) before the generation of 3D cultures. Tumoroids were established in the hydrogel GrowDex ®-T, Nunclon™ Sphera™ flasks, BIOFLOAT™ plates, and Corning ® Elplasia ® plates. Western blot was used to verify antigen protein expression. Hematoxylin-eosin staining was used to evaluate the cell morphology in the 2D and 3D cultures. Mutational analysis of KRAS and EGFR by PCR on extracted DNA from 3D tumoroids generated from cells with known mutations (A549; KRAS G12S mutation, NCI-H1975; EGFR L858R/T790M mutations).

RESULTS: We successfully established 3D cultures from A549, NCI-H1975, HCC-95, and HCC-1588 with all four used cultivation methods. The adenocarcinomas (A549, NCI-H1975) maintained their original IHC features in the tumoroids, while the squamous cell carcinomas (HCC-95, HCC-1588) lost their unique markers in the cultures. PCR analysis confirmed persistent genetic changes where expected.

CONCLUSION: The establishment of tumoroids from lung cancer cell lines is feasible with various methodologies, which is promising for future tumoroid growth from clinical lung cancer samples. However, analysis of relevant markers is a prerequisite and may need to be validated for each model and cell type.

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author
; ; ; ; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Humans, Carcinoma, Non-Small-Cell Lung/pathology, Lung Neoplasms/pathology, Cell Line, Tumor, Proto-Oncogene Proteins p21(ras)/genetics, Cell Culture Techniques/methods, Mutation, ErbB Receptors/genetics, Cell Culture Techniques, Three Dimensional/methods
in
Journal of Cancer Research and Clinical Oncology
volume
150
article number
472
pages
1 - 10
publisher
Springer
external identifiers
  • scopus:85207387384
  • pmid:39441367
ISSN
1432-1335
DOI
10.1007/s00432-024-06003-x
language
English
LU publication?
yes
additional info
© 2024. The Author(s).
id
cf3ccbbc-9a40-4d9a-a109-9b2da14ff69d
date added to LUP
2024-10-26 11:51:15
date last changed
2025-07-01 02:56:15
@article{cf3ccbbc-9a40-4d9a-a109-9b2da14ff69d,
  abstract     = {{<p>PURPOSE: The aim of this study was to set up reliable and reproducible culture conditions for 3D tumoroids derived from non-small cell lung cancer (NSCLC) cell lines to enable greater opportunity for successful cultivation of patient-derived samples.</p><p>METHODS: Four NSCLC cell lines, two adenocarcinomas (A549, NCI-H1975) and two squamous cell carcinomas (HCC-95, HCC-1588), were first cultured in traditional 2D settings. Their expected expression profiles concerning TTF-1, CK7, CK5, and p40 status were confirmed by immunohistochemistry (IHC) before the generation of 3D cultures. Tumoroids were established in the hydrogel GrowDex ®-T, Nunclon™ Sphera™ flasks, BIOFLOAT™ plates, and Corning ® Elplasia ® plates. Western blot was used to verify antigen protein expression. Hematoxylin-eosin staining was used to evaluate the cell morphology in the 2D and 3D cultures. Mutational analysis of KRAS and EGFR by PCR on extracted DNA from 3D tumoroids generated from cells with known mutations (A549; KRAS G12S mutation, NCI-H1975; EGFR L858R/T790M mutations). </p><p>RESULTS: We successfully established 3D cultures from A549, NCI-H1975, HCC-95, and HCC-1588 with all four used cultivation methods. The adenocarcinomas (A549, NCI-H1975) maintained their original IHC features in the tumoroids, while the squamous cell carcinomas (HCC-95, HCC-1588) lost their unique markers in the cultures. PCR analysis confirmed persistent genetic changes where expected.</p><p>CONCLUSION: The establishment of tumoroids from lung cancer cell lines is feasible with various methodologies, which is promising for future tumoroid growth from clinical lung cancer samples. However, analysis of relevant markers is a prerequisite and may need to be validated for each model and cell type.</p>}},
  author       = {{Malmros, Karina and Kirova, Nadi and Kotarsky, Heike and Carlsén, Daniel and Mansour, Mohammed S I and Magnusson, Mattias and Prabhala, Pavan and Brunnström, Hans}},
  issn         = {{1432-1335}},
  keywords     = {{Humans; Carcinoma, Non-Small-Cell Lung/pathology; Lung Neoplasms/pathology; Cell Line, Tumor; Proto-Oncogene Proteins p21(ras)/genetics; Cell Culture Techniques/methods; Mutation; ErbB Receptors/genetics; Cell Culture Techniques, Three Dimensional/methods}},
  language     = {{eng}},
  month        = {{10}},
  pages        = {{1--10}},
  publisher    = {{Springer}},
  series       = {{Journal of Cancer Research and Clinical Oncology}},
  title        = {{3D cultivation of non-small-cell lung cancer cell lines using four different methods}},
  url          = {{http://dx.doi.org/10.1007/s00432-024-06003-x}},
  doi          = {{10.1007/s00432-024-06003-x}},
  volume       = {{150}},
  year         = {{2024}},
}