World J Oncol

World Journal of Oncology, ISSN 1920-4531 print, 1920-454X online, Open Access

Article copyright, the authors; Journal compilation copyright, World J Oncol and Elmer Press Inc

Journal website https://www.wjon.org

Review

Volume 15, Number 2, April 2024, pages 149-168

Pigs: Large Animal Preclinical Cancer Models

Figure 1. Advantages and disadvantages of using small and large preclinical cancer models. Given the similarities between pigs and humans, the pigs lie closer on the scale of translatability to humans compared to small animal models.

Figure 2. Comparative genome viewer alignment comparison between human-pig vs. human-mouse. (a) Comparison between pigs and humans. (b) Comparison between mouse and humans. The colored ribbons represent assembly-to-assembly alignment segments. These are reciprocal best placed alignments by default. Purple represents reverse alignments and green represents forward alignments only. Overall, the comparative genomic viewer shows synteny between the two species that are in comparison. Thicker bands are representative of continuous homology between those species.

Figure 3. Orthologous analysis and comparison between humans, pigs, rats, mice and dogs. (a) Occurrence table presenting a unique number of shared homologous gene clusters among species along with the protein count on the right. (b) Venn diagram showing the number of shared orthologous gene clusters among species along with a bar graph comparison showing the number of orthologous clusters in each species. (c) Pairwise heat map comparison to visualize overlapping cluster numbers for five different species. Our orthologous analysis was conducted using default Orthofinder algorithm, with an e-value of 1 × 10^-2, and inflation value of 1.50.

Figure 4. Expansion and contraction analysis of five species: mice, rats, pigs, dogs, and humans, respectively. In the pie charts, teal indicates expansions while black indicates contractions of orthologous gene clusters.

**Table 1.** Summary of Pig Models
Type of cancer/intervention methods	Method	General comments	References
Glioma/glioblastoma	U87 GM/G-6 (primary culture of GBM)		[16-18]
Spinal cord glioma	Lentiviral vectors	Vector 1: PDGF-B-IRES-eGFP Vector 2: HRAS^G12V-IRES-mPlum Vector 3: pLKO1 backbone + shRNA targeting p53	[18]
Microbrachytherapy	165Ho siloxane particles	U87 cells for tumorigenesis	[19]
Osteosarcoma	Fetal fibroblasts Nuclear and Embryo transfer generating TP53^+/+, TP53^R167H/+, and TP53^R167H/R167H		[20]
	MSCs xenografted AdCre inducible CAG-LSL-TP53^R167H homozygous-KRAS^G12D heterozygous-MYC model		[21]
	MSCs Cre recombinase inducible TP53^R167H followed by somatic cell transfer		[22, 62]
Hematological malignancies	Partial T-cell depleted adult pig bone marrow injected into the intrahepatic portion of the umbilical vein of fetal swine via transuterine injection	Demonstrated chimerism and GVHD MHG-MHC SLAcc (class Ic/IIc)	[23]
	NK cells killed human cancer cell lines (pancreatic, melanoma and CML) in vitro in SCID pig model	Line of Yorkshire pigs that was selected for increased feed efficiency at Iowa State University	[24]
	Naturally occurring CML in MGH MHC pigs		[25]
	HCT (SCF/IL3 mobilized) injected into MGH MHC pigs + total body/thymic irradiation + T-cell depletion + cyclosporine A monotherapy	MGH MHC pigs	[26]
	T- B- NK- SCID pig model by generating ART^-/- IL2RG^-/Y with CRISPR/Cas9 directed mutagenesis		[27]
Colorectal cancer and inflammatory bowel disease	CRISPR/Cas9-mediated excision of the 93-bp fragment containing the ARE and CDE1 element + microinjecting guide RNA plasmids into in vitro fertilized porcine oocytes followed by laparoscopic embryo transfer	TNF^ΔARE pigs	[28]
	Gene-targeted cloned pigs carrying translational stop signals in the APC gene at codons 1061 and 1311		[29]
	CRISPR/Cas9 based LGR5-H2B-GFP model	OLFM4 expression (similar to human small intestine and colon)	[30]
Pancreatic cancer	AdCre CAG-LSL-KRAS^G12D-TP53^R167H into the body of the pancreas or into the pancreatic duct pushed towards the pancreas	Sus scrofa pigs	[31]
	AdCre CAG-LSL-KRAS^G12D-TP53^R167H Oncopig used with core biopsy of pancreas + incubation with AdCre then mixture injected back into pancreas	Oncopigs from University of Missouri National Swine Resource and Research Center	[40]
	Primary porcine fibroblasts with murine Pdx-1 promoter overexpression of oncogene cassette containing MYC, KRAS^G12D and SV40 LT + an inducible negative regulator protein (rtTR-KRAB) via somatic cell nuclear transfer	Gottingen-Ellegaard minipig	[32]
	Transgenic Oncopigs (CAG-KRAS^G12D-IRES-TP53^R167H) Injection Technique 1: Main duct + parenchyma Injection Technique 2: Connecting lobe	Oncopigs from University of Missouri National Swine Resource and Research Center	[33]
Hepatocellular carcinoma	AdCre in vitro expression of KRAS^G12D and TP53^R167H	Minnesota Minipig sire and Yorkshire dams and heterozygous for the transgene	[34, 35]
	Transarterial alcohol injection	Model for cirrhosis development Sus scrofa	[34, 36]
	AdCre KRAS^G12D TP53^R167H Oncopig + CRISPR/Cas9-mediated ko ARID1A + AXIN1	Orthotopic implantation of ko HCC cell in Oncopig model	[37, 38]
Prevention of liver precancerous and cancerous lesions	Model of hereditary tyrosinemia type-1 treatment with in vivo lentiviral vector targeting human fumarylacetoacetate hydrolase transgene	Fumarylacetoacetate hydrolase^-/- via somatic cell transfer	[39]
Immune therapy	Thermal ablation methods	Ex vivo pig livers	[41-43]
	Vaccine-induced peptide-specific cytotoxic T-cell responses	Outbred Danish Landrace/Yorkshire/Duroc pigs	[46]
	Porcine kidney fibroblasts from male Gottingen minipig and transfected with IGH-γ1-γ4 and IGK expression vector. Followed by somatic cell nuclear transfer.	Humanized Gottingen minipigs for toxicological testing of therapeutic recombinant antibodies	[47]
	CRISPR/Cas9 system used to induce deletions within exon 4 of porcine TRDC gene. Electroporation to transfect CRISPR/Cas9 plasmids + Intracytoplasmic microinjection + somatic cell nuclear transfer	TRDC-knockout German Landrace pigs lacking γδ T cells	[48]
	Preclinical testing of immunotherapies for tumor directed cytotoxicity	Oncopig model	[49]
	Autologous hepatocyte derived cell lines transformed using AdCre + transarterial embolization of liver cancer	Oncopig model	[44]
	Xenotransplantation	Oncopig model	[50-53]
	Histotripsy		[45]
Lung cancer Potential interventions for lung cancer	pRosa26-iCas9 pigs by intra-nasal delivery	Chinese Bama mini-pigs	[60]
	Endovascular and percutaneous (biopsy plus incubation with AdCre ex vivo followed by inoculation) delivery		[61]
	Microwave ablation	Oncopigs	[54-56]
	Conventional vs. cone beam CT to monitor microwave ablation		[57]
	Xenogeneic cross-circulation to support human donor		[58, 59]

Table 1. Summary of Pig Models

Type of cancer/intervention methods

Method

General comments

References

Glioma/glioblastoma

U87 GM/G-6 (primary culture of GBM)

[16-18]

Spinal cord glioma

Lentiviral vectors

Vector 1: PDGF-B-IRES-eGFP
Vector 2: HRAS^G12V-IRES-mPlum
Vector 3: pLKO1 backbone + shRNA targeting p53

[18]

Microbrachytherapy

165Ho siloxane particles

U87 cells for tumorigenesis

[19]

Osteosarcoma

Fetal fibroblasts Nuclear and Embryo transfer generating TP53^+/+, TP53^R167H/+, and TP53^R167H/R167H

[20]

MSCs xenografted AdCre inducible CAG-LSL-TP53^R167H homozygous-KRAS^G12D heterozygous-MYC model

[21]

MSCs Cre recombinase inducible TP53^R167H followed by somatic cell transfer

[22, 62]

Hematological malignancies

Partial T-cell depleted adult pig bone marrow injected into the intrahepatic portion of the umbilical vein of fetal swine via transuterine injection

Demonstrated chimerism and GVHD
MHG-MHC SLAcc (class Ic/IIc)

[23]

NK cells killed human cancer cell lines (pancreatic, melanoma and CML) in vitro in SCID pig model

Line of Yorkshire pigs that was selected for increased feed efficiency at Iowa State University

[24]

Naturally occurring CML in MGH MHC pigs

[25]

HCT (SCF/IL3 mobilized) injected into MGH MHC pigs + total body/thymic irradiation + T-cell depletion + cyclosporine A monotherapy

MGH MHC pigs

[26]

T- B- NK- SCID pig model by generating ART^-/- IL2RG^-/Y with CRISPR/Cas9 directed mutagenesis

[27]

Colorectal cancer and inflammatory bowel disease

CRISPR/Cas9-mediated excision of the 93-bp fragment containing the ARE and CDE1 element + microinjecting guide RNA plasmids into in vitro fertilized porcine oocytes followed by laparoscopic embryo transfer

TNF^ΔARE pigs

[28]

Gene-targeted cloned pigs carrying translational stop signals in the APC gene at codons 1061 and 1311

[29]

CRISPR/Cas9 based LGR5-H2B-GFP model

OLFM4 expression (similar to human small intestine and colon)

[30]

Pancreatic cancer

AdCre CAG-LSL-KRAS^G12D-TP53^R167H into the body of the pancreas or into the pancreatic duct pushed towards the pancreas

Sus scrofa pigs

[31]

AdCre CAG-LSL-KRAS^G12D-TP53^R167H Oncopig used with core biopsy of pancreas + incubation with AdCre then mixture injected back into pancreas

Oncopigs from University of Missouri National Swine Resource and Research Center

[40]

Primary porcine fibroblasts with murine Pdx-1 promoter overexpression of oncogene cassette containing MYC, KRAS^G12D and SV40 LT + an inducible negative regulator protein (rtTR-KRAB) via somatic cell nuclear transfer

Gottingen-Ellegaard minipig

[32]

Transgenic Oncopigs (CAG-KRAS^G12D-IRES-TP53^R167H)
Injection Technique 1: Main duct + parenchyma
Injection Technique 2: Connecting lobe

Oncopigs from University of Missouri National Swine Resource and Research Center

[33]

Hepatocellular carcinoma

AdCre in vitro expression of KRAS^G12D and TP53^R167H

Minnesota Minipig sire and Yorkshire dams and heterozygous for the transgene

[34, 35]

Transarterial alcohol injection

Model for cirrhosis development
Sus scrofa

[34, 36]

AdCre KRAS^G12D TP53^R167H Oncopig + CRISPR/Cas9-mediated ko ARID1A + AXIN1

Orthotopic implantation of ko HCC cell in Oncopig model

[37, 38]

Prevention of liver precancerous and cancerous lesions

Model of hereditary tyrosinemia type-1 treatment with in vivo lentiviral vector targeting human fumarylacetoacetate hydrolase transgene

Fumarylacetoacetate hydrolase^-/- via somatic cell transfer

[39]

Immune therapy

Thermal ablation methods

Ex vivo pig livers

[41-43]

Vaccine-induced peptide-specific cytotoxic T-cell responses

Outbred Danish Landrace/Yorkshire/Duroc pigs

[46]

Porcine kidney fibroblasts from male Gottingen minipig and transfected with IGH-γ1-γ4 and IGK expression vector. Followed by somatic cell nuclear transfer.

Humanized Gottingen minipigs for toxicological testing of therapeutic recombinant antibodies

[47]

CRISPR/Cas9 system used to induce deletions within exon 4 of porcine TRDC gene. Electroporation to transfect CRISPR/Cas9 plasmids + Intracytoplasmic microinjection + somatic cell nuclear transfer

TRDC-knockout German Landrace pigs lacking γδ T cells

[48]

Preclinical testing of immunotherapies for tumor directed cytotoxicity

Oncopig model

[49]

Autologous hepatocyte derived cell lines transformed using AdCre + transarterial embolization of liver cancer

Oncopig model

[44]

Xenotransplantation

Oncopig model

[50-53]

Histotripsy

[45]

Lung cancer
Potential interventions for lung cancer

pRosa26-iCas9 pigs by intra-nasal delivery

Chinese Bama mini-pigs

[60]

Endovascular and percutaneous (biopsy plus incubation with AdCre ex vivo followed by inoculation) delivery

[61]

Microwave ablation

Oncopigs

[54-56]

Conventional vs. cone beam CT to monitor microwave ablation

[57]

Xenogeneic cross-circulation to support human donor

[58, 59]