Table 4.

In vitro, 3D culture, and in vivo models used for studying tumor reversion

Model Systems	In vivo/ 3-D / In vitro	Cell Line/Tissue	Observation regarding tumor reversion	Drug Agent/Viral strain	Reference
MSV transformed 3 T3 cell line based Mouse Model	In vivo	Murine Sarcoma Virus transformed mouse 3 T3 cells	Murine sarcoma virus-transformed mouse NIH/3T3 cells (negative for the murine leukemia virus) give rise to a sarcoma virus upon superinfection of murine leukemia virus. The revertants support leukemia virus growth and show enhanced sensitivity to murine sarcoma superinfection and, like normal cells, do not release RNA-dependent DNA polymerase activity.	Murine Leukemia Virus (MuLV)	Fischinger et al 1972, [144]
Twenty four acute promyelocytic leukemia (APL) patients	In vivo	Human APL	APL patients treated with all-trans retinoic acid attained complete remission without developing bone marrow hypoplasia, and a gradual terminal differentiation in bone marrow derived tumor cells was observed as evident due to presence of Auer rods in mature granulocytes, followed by the re-emergence of normal hematopoietic cells upon remission.	Retinoic Acid (45 to 100 mg/m2/day)	Huang et al 1988, [145]; Zhou et al 2007, [146]
Transgenic Mice Model (MTA transgene)	In vivo	Mice carrying the tetracyclineresponsive trans activator (tTA) gene (MTA transgene)	Controlled expression of simian virus 40 T antigen (Tag) in the submandibular gland of transgenic mice four months from the time of birth induced cellular transformation and extensive ductal hyperplasia. Silencing of Tag led to reversal of hyperplasia for 3 weeks, but NOT after seven months as hyperplasia persistence was observed. Reversal of ploidy in ductal cells was possible in 4 months old animals, and observed to remains polyploidy at the age of 7 months even when devoid of TAg.	Simian Virus 40 (SV40)	Ewald et al 1996, [147]
BCR–ABL1-(tTA) induced Double Transgenic Mice Model	In vivo	Double transgenic mice (BCR–ABL1-tetracycline transactivator (tTA))	Withdrawal of tetracycline administration from double transgenic animals (BCR–ABL1-tetracycline transactivator (tTA) permits BCR-ABL1 expression and cause lethal leukemia. The rapid disappearance of leukemic cells from the blood suggested apoptosis rather than differentiation as the underlying mechanism for reversing the phenotype in this model. A constitutive BCR–ABL1 expression is crucial for maintaining the cancer of hematopoietic system and phenotype of the leukemic cells is completely reversible at the advanced stages of the disease.	Withdrawal of Tetracycline	Huettner et al 2000, [148]
Polyoma transformed hamster embryo cell model	In vivo	Hamster embryo cells (transformed by polyoma virus inoculated with large plaque virus LPll)	Hamster embryonic cell transformed using polyomavirus after inoculating with large virus LP-II plaque. A high frequency of variants with reversion phenotype observed in transformed cells.	Carmine	Rabinowitz et al 1968, [149]
MGI (macrophages and granulocyte inducer) grafted mice model	In vivo	Human and mice myleoid leukemia cells	Chromosomal studies of MGI + D+ leukemic cells showed that all the chromosomes could still be persuaded to develop a normal differentiation phenotype without a completely normal complement. Reversion of the phenotype of malignant cells with standard growth control without a completely normal chromosome complement was observed in other cell types as well.	Grafting of MGI producing cells	Sachs et al 1978, [150]
SCID Mice for making tumors derived from K562 and KS cells	In vivo	K562 leukemia cells and the clone KS resistant to cytopathic effect of H1 parvovirus	H-1 parvovirus preferentially kills neoplastic cells, and therefore used for selecting cells with the suppressed phenotype. KS (a cell line derived from K562) resistan to cytopathic effect of the H-1 virus displays a suppressed malignant phenotype. This and cellular resistance to H-1 killing appear to depend on the activity of wild-type p53.	H-1 parvovirus	Telerman et al 1993 [151]
3D In vitro Culture system ‘on top’ and disease-on-achip (DOC)	In vitro, DOC model to mimic TME.	Pre-invasive (S2 cells) and invasive (T4–2 and MDA-MB-231 Cell line)	The DOC model used for the breast cancer cells with varied tumorigenecity. In the 3D culture DOC model to mimic TME, the complex treatment had non-toxic effects on S2 cells, but induce significant cytotoxicity in invasive cells. Also, in 3D model, the cells were able to produce breast ductal architecture. Treatment also abolished tumor phenotype of invasive cells downregulation of markers such as EGFR, P50, NFκB and β1-integrin was observed.	e trans-[Ru(PPh3) 2(N,Ndimethyl-Nthiophenylt hioureatok2O, S)(bipy)]PF6 complex	Becceneri et al 2020 [152]
3D culture model to test RADARADARADARADACONH2 (RADA16)	In vitro, Effect of RADA on CD44+/ CD24-cells derived from breast cancer cell line	CD44+/CD24-sub population selected after sub-culturing MDA-MB-435S	MDA-MB-435S was enriched in CD44+/CD24-phenotype expressing cells. As compared with matrigel and collagen I, cells cultured in 3D RADA16 nano-fiber scaffold showed reversion of tumor phenotype, and formed round colonies and well-organized centric nucleus with regular morphology of the cells.	Cancer cells tested by culturing in 3D model either in RADA16 nanofiber scaffold versus Matrigel collagen I	Mi et al 2015 [153]
3D model using Engelbreth-Holm-Swarm extracellular matrix extract (EHS) matrigel and rat-tail collagen	In vitro, Cells embedded in the EHS Matrix as single cells allowed to grow for 10–121 days	Human breast epithelial cell line (S1), and invasive (T4–2 cell line)	Blocking of the β1-integrin using anti- β1-integrin MAB (clone AIIB2), T4–2 cells resistant to the antibody, but developed morphology very much alike S1 cells.	anti- β1-integrin MAB	Weaver et al 1997 [154]
3D laminin-rich basement membrane (3DlrBM) model for studying human breast cancer	In vitro	Breast (MDA-MD-231), and cervical cancer cell line (HeLa)	The retroviral expression transferred to re-establish HoxD10 expression in the malignant breast tumor cells. A phenotypic reversion after decrease in the expression levels of α3 integrin was observed along with decelerated cellular proliferation.	Retroviral gene transfer to restore HoxD10 expression	Carrio et al 2005 [25]
3D culture system for studying human breast cancer	In vitro	Epithelial cell line (HMT-3522), and breast cancer cell line (T4–2)	Downregulation of EGFR and β1-integrin observed in breast tumors and normal cell line upon treatment with an antibody against β1-integrin function-blocking mAb. This further led to growth arrest and tumor phenotype changes, looking more like normal breast cell morphology.	β1-integrin function blocking mAb	Wang et al 1998 [155]
Biodegradable meshwork (Hyalograft 3D): primary breast cancer cells cultured in 3D collagen-I gels both as mono- and as co-culture with human mammary fibroblasts (HMFs).	In vitro	PBCs and HMFs were used in the ratio of 1:2. Primary breast carcinoma cells	In a total of 38% of the cases, reverted tumor phenotype was observed. Presence of acini formation was observed as a conversion characteristic into the normal phenotype, which was reported to be 2–7 fold, and glandular structures were observed in reverted co-cultured cells. In the isolated primary breast carcinoma cells; out-of 13, only 5 exhibited reversion of their malignant phenotype. Also, differentially expressed genes were identified such as ELF5, MAL, SQLE, MAP6, and ZMYND11.	PBC cultured alone or with HMFs for comparative analysis	Romer et al 2013 [156]
3D basement membrane culture model	In vitro	Mammary epithelial cells (MCF-10A cell lines)	RNAi mediated inhibition of Bim expression blocks the luminal apoptosis and slow down the formation of lumen.	RNAi to block Bim expression	Reginato et al 2005 [157]
Human breast 3D tissue morphogenesis models	In vitro	Human breast cancer MCF7 cells	MCF7 cells were co-cultured with primary human breast fibroblasts. The presence of normal breast fibroblasts constitutes the minimal permissive microenvironment to induce near-complete tumor phenotypic reversion.	Cells alone or cocultured with primary human breast fibroblast cells	Krause et al 2010 [158]
3DlrECM	In vitro	Breast cancer cell line T4–2	Small molecule inhibitor of TACE, TAPI-2, reverted the malignant phenotype of T4–2 cells into phenotypically normal mammary acinus like architectures. The TACE-dependent shedding of amphiregulin and TGF-α was also observed in several additional breast cancer cell lines.	Small molecule inhibitor of TACE, TAPI-2	Kenny et al 2007 [159]
Kirsten sarcoma virustransformed NIH/3T3 cell line model	In vitro	NIH/3T3, DT (Ki/HGPRT- NIH/3T3), Ki/TK- NIH	Flat revertants with in vivo reduced tumorigenicity isolated from populations of 3 T3 cells transfected with a cDNA expression library derived from normal human fibroblasts.	Kirsten sarcoma virus	Noda et al 1989, [160]
Lysosomal-type sialidase b16 melanoma cells Murine model	In vitro	B16-BL6 murine melanoma cells	Lysosomal sialidase overexpression inhibits the metastatic potential of B16 melanoma, at least partially through reduction of cell growth and sensitization to apoptosis. This shows sialidase is involved in cellular functions, which are affecting malignancy characteristics of cancer cells.	Lysosomal-type sialidase	Kato et al 2001, [108]
GM3-mediated cell line model	In vitro	KK47 (noninvasive & nonmetastatic) and YTS1 (highly invasive and metastatic)	Ganglioside GM3 expression was higher in KK47 than in YTS1 cells. GM3 shows multiple functions like Integrin α3 with CD9 have more vital interaction. RNAi was used to knockdown CD9 that gives high cell motility. An addition of GM3 exogenously induces reversion of high motility YTS1 to low motility phenotype. An increased level of GM3 suppresses the motility as well as invasiveness of the tumor cells.	Gangliosid es GM3	Mitsuzuka et al 2005, [161]
Viral oncoprotein Jun (v-Jun) Fibroblast cell line model	In vitro	Mouse fibroblast cell line C3H 10 T1/2 & the chicken fibroblast cell line DF1	Upregulation of GM3 synthase occurs upon GM3 transfection in v-Jun-transformed 10 T1/2 cells, and this reverts the oncogenic phenotype into normal as indicated by anchorage-independent growth.	Sialosyllac tosylcera mide	Miura et al 2004, [162]
Human breast epithelial cells	In vitro	MCF10A Treated with or without PD032590 (MEK inhibitor)	MCF10A treatment with MEKi showed phenotypic reversion as well as downregulation of different molecules involved in membrane transport, metabolism, cell adhesion, & downregulation of biological processes crucial for tumor-related phenotypes including cellular proliferation & metastasis	MEK Inhibitior	Leung et al 2020, [28]