Abstract
The growth of human tumor cells transplanted into immunodeficient mice is frequently studied to gain understanding about the way potential drug treatments interfere with growth in vivo. A wide range of methods is available for learning about specific aspects of tumor cell behavior, for example, cells may be administered to follow their ability to grow close to the site of injection which may be at a generic site or one specific to that type of tumor. Some models of metastasis follow the appearance of a tumor mass after intravascular administration of tumor cells; others score remote growth after removal of a primary tumor implanted subcutaneously. Assessing metastatic growth may increasingly rely on serial observation of tumor cell numbers as seen by whole-body imaging, but the sensitivity of these methods is poor in terms of the minimum number of cells detectable, and histological follow-up to establish tumor cell numbers can be confounded by variable expression or even silencing of reporter genes. Here we describe how fluorescence in situ hybridization (FISH) using commercially available probes can very easily be used to detect even single metastatic tumor cells in mouse models, using routinely fixed and processed tissue samples, and without the tumor cell lines needing to express engineered reporter genes. The FISH protocol can be combined with other standard histological protocols to study the behavior of tumor cells and adjacent host cells to improve our understanding of tumor–stroma interactions, and is also useful for simultaneous demonstration of the cell of origin and phenotype of cells used in regenerative medicine-based applications.
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References
O’Neill K, Lyons SK, Gallagher WM, Curran KM, Byrne AT (2010) Bioluminescent imaging: a critical tool in pre-clinical oncology research. J Pathol 220:317–327
Cao YA, Wagers AJ, Beilhack A, Dusich J, Bachmann MH, Negrin RS, Weissman IL, Contag CH (2004) Shifting foci of hematopoiesis during reconstitution from single stem cells. Proc Natl Acad Sci USA 101:221–226
Oh S, Stish BJ, Sachdev D, Chen H, Dudek AZ, Vallera DA (2009) A novel reduced immunogenicity bispecific targeted toxin simultaneously recognizing human epidermal growth factor and interleukin-4 receptors in a mouse model of metastatic breast carcinoma. Clin Cancer Res 15:6137–6147
Lyons SK, Lim E, Clermont AO, Dusich J, Zhu L, Campbell KD, Coffee RJ, Grass DS, Hunter J, Purchio T, Jenkins D (2006) Noninvasive bioluminescence imaging of normal and spontaneously transformed prostate tissue in mice. Cancer Res 66:4701–4707
Leithauser F, Trobonjaca Z, Reimann J, Moller P (2001) In situ characterization of genetically targeted (green fluorescent) single cells and their microenvironment in an adoptive host. Am J Pathol 158:1975–1983
Brittan M, Braun KM, Reynolds LE, Conti FJ, Reynolds AR, Poulsom R, Alison MR, Wright NA, Hodivala-Dilke KM (2005) Bone marrow cells engraft within the epidermis and proliferate in vivo with no evidence of cell fusion. J Pathol 205:1–13
Fang TC, Otto WR, Rao J, Jeffery R, Hunt T, Alison MR, Cook HT, Wright NA, Poulsom R (2008) Haematopoietic lineage-committed bone marrow cells, but not cloned cultured mesenchymal stem cells, contribute to regeneration of renal tubular epithelium after HgCl2-induced acute tubular injury. Cell Prolif 41:575–591
Zhang SC, Wernig M, Duncan ID, Brustle O, Thomson JA (2001) In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat Biotechnol 19: 1129–1133
Burghoff S, Ding Z, Godecke S, Assmann A, Wirrwar A, Buchholz D, Sergeeva O, Leurs C, Hanenberg H, Muller HW, Bloch W, Schrader J (2008) Horizontal gene transfer from human endothelial cells to rat cardiomyocytes after intracoronary transplantation. Cardiovasc Res 77:534–543
Gao J, Tombal B, Isaacs JT (1999) Rapid in situ hybridization technique for detecting malignant mouse cell contamination in human xenograft tissue from nude mice and in vitro cultures from such xenografts. Prostate 39:67–70
Brustle O, Choudhary K, Karram K, Huttner A, Murray K, Dubois-Dalcq M, McKay RD (1998) Chimeric brains generated by intraventricular transplantation of fetal human brain cells into embryonic rats. Nat Biotechnol 16:1040–1044
Speel EJ, Herbergs J, Ramaekers FC, Hopman AH (1994) Combined immunocytochemistry and fluorescence in situ hybridization for simultaneous tricolor detection of cell cycle, genomic, and phenotypic parameters of tumor cells. J Histochem Cytochem 42:961–966
Speel EJ, Jansen MP, Ramaekers FC, Hopman AH (1994) A novel triple-color detection procedure for brightfield microscopy, combining in situ hybridization with immunocytochemistry. J Histochem Cytochem 42: 1299–1307
Mueller M, Wacker K, Hickey WF, Ringelstein EB, Kiefer R (2000) Co-localization of multiple antigens and specific DNA. A novel method using methyl methacrylate-embedded semithin serial sections and catalyzed reporter deposition. Am J Pathol 157:1829–1838
Tubbs RR, Pettay J, Roche P, Stoler MH, Jenkins R, Myles J, Grogan T (2000) Concomitant oncoprotein detection with fluorescence in situ hybridization (CODFISH): a fluorescence-based assay enabling simultaneous visualization of gene amplification and encoded protein expression. J Mol Diagn 2:78–83
Dundas SR, Boyle S, Bellamy CO, Hawkins W, Garden OJ, Ross JA, Bickmore W (2001) Dual Y-chromosome painting and immunofluorescence staining of archival human liver transplant biopsies. J Histochem Cytochem 49:1321–1322
Martin-Subero JI, Chudoba I, Harder L, Gesk S, Grote W, Novo FJ, Calasanz MJ, Siebert R (2002) Multicolor-FICTION: expanding the possibilities of combined morphologic, immunophenotypic, and genetic single cell analyses. Am J Pathol 161:413–420
Khosrotehrani K, Stroh H, Bianchi DW, Johnson KL (2003) Combined FISH and immunolabeling on paraffin-embedded tissue sections for the study of microchimerism. Biotechniques 34:242–244
Pezzolo A, Parodi F, Corrias MV, Cinti R, Gambini C, Pistoia V (2007) Tumor origin of endothelial cells in human neuroblastoma. J Clin Oncol 25:376–383
Steel JH, O’Donoghue K, Kennea NL, Sullivan MH, Edwards AD (2005) Maternal origin of inflammatory leukocytes in preterm fetal membranes, shown by fluorescence in situ hybridisation. Placenta 26:672–677
Choolani M, O’Donnell H, Campagnoli C, Kumar S, Roberts I, Bennett PR, Fisk NM (2001) Simultaneous fetal cell identification and diagnosis by epsilon-globin chain immunophenotyping and chromosomal fluorescence in situ hybridization. Blood 98:554–557
Johnson KL, Zhen DK, Bianchi DW (2000) The use of fluorescence in situ hybridization (FISH) on paraffin-embedded tissue sections for the study of microchimerism. Biotechniques 29:1220–1224
Poulsom R, Forbes SJ, Hodivala-Dilke K, Ryan E, Wyles S, Navaratnarasah S, Jeffery R, Hunt T, Alison M, Cook T, Pusey C, Wright NA (2001) Bone marrow contributes to renal parenchymal turnover and regeneration. J Pathol 195:229–235
Direkze NC, Forbes SJ, Brittan M, Hunt T, Jeffery R, Preston SL, Poulsom R, Hodivala-Dilke K, Alison MR, Wright NA (2003) Multiple organ engraftment by bone-marrow-derived myofibroblasts and fibroblasts in bone-marrow-transplanted mice. Stem Cells 21: 514–520
Ferlicot S, Vernochet A, Romana S, Ortin-Serrano M, Letierce A, Bregerie O, Durrbach A, Guettier C (2010) Microchimerism in renal allografts: clinicopathological associations according to the type of chimeric cells. Histopathology 56:188–197
Revoltella RP, Papini S, Rosellini A, Michelini M, Franceschini V, Ciorba A, Bertolaso L, Magosso S, Hatzopoulos S, Lorito G, Giordano P, Simoni E, Ognio E, Cilli M, Saccardi R, Urbani S, Jeffery R, Poulsom R, Martini A (2008) Cochlear repair by transplantation of human cord blood CD133+ cells to nod-scid mice made deaf with kanamycin and noise. Cell Transplant 17:665–678
Franceschini V, Bettini S, Pifferi S, Rosellini A, Menini A, Saccardi R, Ognio E, Jeffery R, Poulsom R, Revoltella RP (2009) Human cord blood CD133+ stem cells transplanted to nod-scid mice provide conditions for regeneration of olfactory neuroepithelium after permanent damage induced by dichlobenil. Stem Cells 27:825–835
Laflamme MA, Chen KY, Naumova AV, Muskheli V, Fugate JA, Dupras SK, Reinecke H, Xu C, Hassanipour M, Police S, O’Sullivan C, Collins L, Chen Y, Minami E, Gill EA, Ueno S, Yuan C, Gold J, Murry CE (2007) Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts. Nat Biotechnol 25:1015–1024
Laakso M, Tanner M, Isola J (2006) Dual-colour chromogenic in situ hybridization for testing of HER-2 oncogene amplification in archival breast tumours. J Pathol 210:3–9
Hollinshead M, Sanderson J, Vaux DJ (1998) Vector alkaline phophatase substrate Blue III: one substrate for brightfield histochemistry and high-resolution fluorescence imaging by confocal laser scanning microscopy. Histochem J 30:577–581
Speel EJ, Schutte B, Wiegant J, Ramaekers FC, Hopman AH (1992) A novel fluorescence detection method for in situ hybridization, based on the alkaline phosphatase-fast red reaction. J Histochem Cytochem 40:1299–1308
Chin SF, Daigo Y, Huang HE, Iyer NG, Callagy G, Kranjac T, Gonzalez M, Sangan T, Earl H, Caldas C (2003) A simple and reliable pretreatment protocol facilitates fluorescent in situ hybridisation on tissue microarrays of paraffin wax embedded tumour samples. Mol Pathol 56:275–279
Yen TH, Alison MR, Cook HT, Jeffery R, Otto WR, Wright NA, Poulsom R (2007) The cellular origin and proliferative status of regenerating renal parenchyma after mercuric chloride damage and erythropoietin treatment. Cell Prolif 40:143–156
Pardo OE, Latigo J, Jeffery RE, Nye E, Poulsom R, Spencer-Dene B, Lemoine NR, Stamp GW, Aboagye EO, Seckl MJ (2009) The fibroblast growth factor receptor inhibitor PD173074 blocks small cell lung cancer growth in vitro and in vivo. Cancer Res 69:8645–8651
Acknowledgements
We are grateful to our colleagues Dr Adam Giangreco and Dr Olivier Pardo for providing some of the tissues illustrated. This work was supported by Cancer Research UK.
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Jeffery, R., Seedhar, P., Poulsom, R. (2014). Identifying the Origin and Phenotype of Cells in Tumor Xenografts. In: Dwek, M., Schumacher, U., Brooks, S. (eds) Metastasis Research Protocols. Methods in Molecular Biology, vol 1070. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4614-8244-4_18
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DOI: https://doi.org/10.1007/978-1-4614-8244-4_18
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