The role of mouse models in colorectal cancer research—The need and the importance of the orthotopic models

Colorectal cancer is a worldwide health burden,with high incidence and mortality,especially in the advanced stages of the disease.Preclinical models are very important and valuable to discover and validate early and specific biomarkers as well as new therapeutic targets.In order to accomplish that,the animal models must replicate the clinical evolution of the disease in all of its phases.In this article,we review the existent mouse models,with their strengths and weaknesses in the replication of human cancer disease progression,with major focus on orthotopic models.

Evaluation of direct intramural injection to the bladder wall as a method for developing orthotopic tumor models

Background:Bladder cancer poses a great burden on society and its high rate of recurrence and treatment failure necessitates use of appropriate animal models to study its pathogenesis and test novel treatments.Orthotopic models are superior to other types since they provide a normal microenvironment.Four methods are described for developing bladder cancer models inside the animal’s bladder.Direct intramural injection is one of these methods and is widely used.However,its efficacy in model development has not yet been studied.We aimed to evaluate the efficacy and success rate of the direct intramural injection method of developing an orthotopic model for the study of bladder cancer.Method:Tumor cell lines were prepared in four microtubes.Aliquots of 200×10^(3) cells were injected through a 27 gauge needle into the ventral wall of the bladders of 4male and 4 female BALB/c mice following a midline 1 cm laparotomy incision.In addition,1 million cells from each microtube were injected into the flanks of control mice.To prevent infection and alleviate pain,5 mg/kg enrofloxacin and 2.5 mg/kg flunixin meglumine,respectively,were injected subcutaneously.Results:Tumors formed in all mice,resulting in 100% take rate and zero post-operation mortality.Surgery time was≤15 min per mouse.In two mice,tumors were found in the peritoneal space as well.Conclusion:Direct intramural injection is a rapid,reliable,and reproducible method for developing orthotopic models of bladder cancer.It can be done on both male and female mice and only requires readily available surgical tools.However,needle track can result in cell spillage and peritoneal tumors.

Establishment of image-guided radiotherapy of orthotopic hepatocellular carcinoma mouse model

Background:Hepatocellular carcinoma(HCC)is the most common type of liver cancer.Recently,developments in radiotherapy technology have led to radiotherapy becoming one of the main therapeutics of HCC.Therefore,a suitable animal model for radiotherapy of the orthotopic HCC mouse model is urgently needed.Methods:In the present study,Hepa1-6 cells were injected into the liver of C57BL/6 mice in situ to mimic the pathological characteristics of the original HCC.Tumor formation was monitored by applying magnetic resonance imaging techniques and verified by H&E histopathological staining,AFP staining,and Ki67 staining.A single dose of 10 Gy X-ray was applied to simulate clinical radiotherapy plans using image-guided radiotherapy(IGRT)equipment.The efficiency of radiotherapy was then assessed by examining tumor size and weight one week after radiation.Cleaved-caspase3 staining and TUNEL were used to assess apoptosis in tumor tissues.Results:Intrahepatic tumor development was detected in the liver according using MRI.A high-density shadow could be seen 10 days after cell injection,which indicated the formation of HCC in vivo.The tumors grew steadily bigger,and underwent precision radiotherapy 20 days after injection.The typical pathological characteristics of HCC,such as large,deeply stained nuclei and irregular cell size,were visible with H&E staining.After radiotherapy,significantly higher expression of the immunohistochemical markers Ki67 and AFP were detected in tumor tissue than in the nearby normal tissue.Compared with the control group,the tumor volume(p=0.05)and weight(p<0.05)of the irradiated group were significantly reduced.In addition,a higher frequency of apoptosis was identified in irradiated HCC tumor tissue using the TUNEL and cleaved-caspase3 staining assay.Conclusions:In a well-established orthotopic HCC model,MRI was utilized to monitor the formation of tumors,and IGRT was used to simulate clinical radiotherapy.The present study could provide a suitable preclinical system for HCC radiotherapyrelated studies.

Establishment of an orthotopic transplantation tumor model of hepatocellular carcinoma in mice

AIM:To improve the outcome of orthotopic transplantation in a mouse model,we used an absorbable gelatin sponge(AGS) in nude mice to establish an orthotopic implantation tumor model.METHODS:MHCC-97L hepatocellular carcinoma(HCC)cells stably expressing the luciferase gene were injected into the subcutaneous region of nude mice.One week later,the ectopic tumors were harvested and transplanted into the left liver lobe of nude mice.The AGS was used to establish the nude mouse orthotopic implantation tumor model.The tumor suppressor gene,paired box gene 5(PAX5),which is a tumor suppressor in HCC,was transfected into HCC cells to validate the model.Tumor growth was measured by bioluminescence imaging technology.Semi-quantitative reverse transcription polymerase chain reaction(RT-PCR) and histopathology were used to confirm the tumorigenicity of the implanted tumor from the MHCC-97L cell line.RESULTS:We successfully developed an orthotopic transplantation tumor model in nude mice with the use of an AGS.The success rate of tumor transplantation was improved from 60% in the control group to 100% in the experimental group using AGS.The detection of fluorescent signals showed that tumors grew in all live nude mice.The mice were divided into 3 groups:AGS-,AGS+/PAX5-and AGS+/PAX5 +.Tumor size was significantly smaller in PAX5 transfected nude mice compared to control mice(P < 0.0001).These fluorescent signal results were consistent with observations made during surgery.Pathologic examination further confirmed that the tissues from the ectopic tumor were HCC.Results from RT-PCR proved that the HCC originated from MHCC-97L cells.CONCLUSION:Using an AGS is a convenient and efficient way of establishing an indirect orthotopic liver transplantation tumor model with a high success rate.

Establishment of an orthotopic pancreatic cancer mouse model: Cells suspended and injected in Matrigel

AIM: To establish an orthotopic mouse model of pancreatic cancer that mimics the pathological features of exocrine pancreatic adenocarcinoma.

Inhibition of growth and metastasis of triple-negative breast cancer targeted by Traditional Chinese Medicine Tubeimu in orthotopic mice models

Objective： Triple-negative breast cancer（TNBC） is highly invasive and metastatic, which is in urgent need of transformative therapeutics. Tubeimu（TBM）, the rhizome of Bolbostemma paniculatum（Maxim.） Franquet, is one of the Chinese medicinal herbs used for breast diseases since the ancient times. The present study evaluated the efficacy, especially the anti-metastatic effects of the dichloromethane extract of Tubeimu（ETBM） on TNBC orthotopic mouse models and cell lines.Methods： We applied real-time imaging on florescent orthotopic TNBC mice model and tested cell migration and invasion abilities with MDA-MB-231 cell line. Digital gene expression sequencing was performed and Kyoto Encyclopedia of Genes and Genomes（KEGG） analysis applied to explore the pathways influenced by ETBM.Moreover, quantitative real-time polymerase chain reactions（q RT-PCR） and Western blot were delivered to confirm the gene expression changes.Results： ETBM exhibited noticeable control on tumor metastasis and growth of TNBC tumors with no obvious toxicity. In compliance with this, it also showed inhibition of cell migration and invasion in vitro. Its impact on the changed biological behavior in TNBC may be a result of decreased expression of integrin β1（ITGβ1）, integrin β8（ITGβ8） and Rho GTPase activating protein 5（ARHGAP5）, which disabled the focal adhesion pathway and caused change in cell morphology.Conclusions： This study reveals that ETBM has anti-metastatic effects on MDA-MB-231-GFP tumor and may lead to a new therapeutic agent for the integrative treatment of highly invasive TNBC.

Mouse models of epithelial ovarian cancer for preclinical studies

Epithelial ovarian cancer(EOC) is the leading cause of gynecological cancer-related mortality in the developed world. EOC is a heterogeneous disease represented by several histological and molecular subtypes. Therefore, exploration of relevant preclinical animal models that consider the heterogenic nature of EOC is of great importance for the development of novel therapeutic strategies that can be translated clinically to combat this devastating disease. In this review, we discuss recent progress in the development of preclinical mouse models for EOC study as well as their advantages and limitations.

Uncoupling protein 2 deficiency of non-cancerous tissues inhibits the progression of pancreatic cancer in mice

Background: Pancreatic ductal adenocarcinoma(PDAC) is a disease of the elderly mostly because its development from preneoplastic lesions depends on the accumulation of gene mutations and epigenetic alterations over time. How aging of non-cancerous tissues of the host affects tumor progression, however, remains largely unknown. Methods: We took advantage of a model of accelerated aging, uncoupling protein 2-deficient( Ucp2 knockout, Ucp2 KO) mice, to investigate the growth of orthotopically transplanted Ucp2 wild-type(WT) PDAC cells(cell lines Panc02 and 6606PDA) in vivo and to study strain-dependent differences of the PDAC microenvironment. Results: Measurements of tumor weights and quantification of proliferating cells indicated a significant growth advantage of Panc02 and 6606PDA cells in WT mice compared to Ucp2 KO mice. In tumors in the knockout strain, higher levels of interferon-γ m RNA despite similar numbers of tumor-infiltrating T cells were observed. 6606PDA cells triggered a stronger stromal reaction in Ucp2 KO mice than in WT animals. Accordingly, pancreatic stellate cells from Ucp2 KO mice proliferated at a higher rate than cells of the WT strain when they were incubated with conditioned media from PDAC cells. Conclusions: Ucp2 modulates PDAC microenvironment in a way that favors tumor progression and implicates an altered stromal response as one of the underlying mechanisms.

Anti-tumor effects of polybutylcyanoacrylate nanoparticles of diallyl trisulfide on orthotopic transplantation tumor model of hepatocellular carcinoma in BALB/c nude mice

Background Hepatocellular carcinoma （HCC） ranked the second among the causes of cancer mortality in China since the 1990s. Up to now, medication still plays an important role in the treatment of HCC. The therapies based on the allicin as a potential chemopreventive analog although is in its infancy at the present time, may have a significant role in the future management of HCC. Diallyl trisulfide （DATS） is a natural compound derived from garlic. In this study, we investigated the inhibitory effects of hepatic targeted polybutylcyanoacrylate nanoparticles of diallyl trisulfide （DATS-PBCA-NP） on orthotopic transplanted HepG2 hepatocellular carcinoma in nude mice. Methods DATS-PBCA-NP were detected by transmission electron microscope （TEM） and high-performance liquid chromatography （HPLC）. The orthotopic transplantation HCC models were established by implanting HCC HepG2 xenograft bits under the envelope of the mice liver. Successful models （n=29） were divided into 4 groups： normal saline （NS）, empty nanoparticles （EN）, DATS and DATS-PBCA-NP were intravenously administered to the mice respectively for 2 weeks. In vivo antitumor efficacy was evaluated by the measurement of tumor volume. Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling （TUNEL） assay and protein levels of apoptosis and cell proliferation proteins by immunoblotting in tumor tissues were performed to elucidate the possible mechanism. Results DATS-PBCA-NP possessed smooth and round appearance, dispersed well, and released in vitro in accord with double phase kinetics model. DATS-PBCA-NP changed the tissue/organ distribution of DATS in vivo. The successful rate of tumor implantation was 100%. Intravenous administration of DATS-PBCA-NP significantly retarded the growth of orthotopically transplanted hepatoma in BALB/c nude mice （compared with the other three groups, all P〈0.05） without causing weight loss （P〉0.05）. TUNEL staining showed that the tumors from DATS-PBCA-NP treated mice exhibited a markedly higher apoptotic index compared with control tumors. Western blot analysis of tumor tissue revealed that the down-regulated expression of proliferation cell nuclear antigen （PCNA） and Bcl-2 proteins in DATS-PBCA-NP group, and there were no significant differences in the expression of Fas, FasL and Bax proteins among the four groups （P〉0.05）. Conclusions DATS-PBCA-NP has good prolonged release effect in vivo and hepatic-targeted activity, and significant anti,tumor effect on the orthotopic transplantation HCC model in mice in association with the suppression of proliferation and the induction of apoptosis of tumor cells. These advantages are probably due to their liver targeting characteristics and consequently bring a higher anti-tumor activity.

A cadaveric breast cancer tissue phantom for phase-contrast X-ray imaging applications

Background:As mammography X-ray imaging technologies advance and provide elevated contrast in soft tissues,a need has developed for reliable imaging phantoms for use in system design and component calibration.In advanced imaging modalities such as refraction-based methods,it is critical that developed phantoms capture the biological details seen in clinical precancerous and cancerous cases while minimizing artifacts that may be caused due to phantom production.This work presents the fabrication of a breast tissue imaging phantom from cadaveric breast tissue suitable for use in both transmission and refraction-enhanced imaging systems.Methods:Human cancer cell tumors were grown orthotopically in nude athymic mice and implanted into the fixed tissue while maintaining the native tumor/adipose tissue interface.Results:The resulting human–murine tissue hybrid phantom was mounted on a clear acrylic housing for absorption and refraction X-ray imaging.Digital breast tomosynthesis was also performed.Conclusion:Both attenuation-based imaging and refraction-based imaging of the phantom are presented to confirm the suitability of this phantom's use in both imaging modalities.

Rescue of p53 functions by in vitro-transcribed mRNA impedes the growth of high-grade serous ovarian cancer

Background:The cellular tumor protein p53(TP53)is a tumor suppressor gene that is frequently mutated in human cancers.Among various cancer types,the very aggressive high-grade serous ovarian carcinoma(HGSOC)exhibits the high-est prevalence of TP53 mutations,present in>96%of cases.Despite intensive efforts to reactivate p53,no clinical drug has been approved to rescue p53 func-tion.In this study,our primary objective was to administer in vitro-transcribed(IVT)wild-type(WT)p53-mRNA to HGSOC cell lines,primary cells,and ortho-topic mouse models,with the aim of exploring its impact on inhibiting tumor growth and dissemination,both in vitro and in vivo.Methods:To restore the activity of p53,WT p53 was exogenously expressed in HGSOC cell lines using a mammalian vector system.Moreover,IVT WT p53 mRNA was delivered into different HGSOC model systems(primary cells and patient-derived organoids)using liposomes and studied for proliferation,cell cycle progression,apoptosis,colony formation,and chromosomal instabil-ity.Transcriptomic alterations induced by p53 mRNA were analyzed using RNA sequencing in OVCAR-8 and primary HGSOC cells,followed by ingenuity path-way analysis.In vivo effects on tumor growth and metastasis were studied using orthotopic xenografts and metastatic intraperitoneal mouse models.Results:Reactivation of the TP53 tumor suppressor gene was explored in differ-ent HGSOC model systems using newly designed IVT mRNA-based methods.The introduction of WT p53 mRNA triggered dose-dependent apoptosis,cell cycle arrest,and potent long-lasting inhibition of HGSOC cell proliferation.Transcriptome analysis of OVCAR-8 cells upon mRNA-based p53 reactivation revealed significant alterations in gene expression related to p53 signaling,such as apoptosis,cell cycle regulation,and DNA damage.Restoring p53 function concurrently reduces chromosomal instability within the HGSOC cells,under-scoring its crucial contribution in safeguarding genomic integrity by moderating the baseline occurrence of double-strand breaks arising from replication stress.Furthermore,in various mouse models,treatment with p53 mRNA reduced tumor growth and inhibited tumor cell dissemination in the peritoneal cavity in a dose-dependent manner.Conclusions:The IVT mRNA-based reactivation of p53 holds promise as a potential therapeutic strategy for HGSOC,providing valuable insights into the molecular mechanisms underlying p53 function and its relevance in ovarian cancer treatment.

The associated killing of hepatoma cells using multilayer drug-loaded mats combined with fast neutron therapy

Chemotherapeutic and radiation therapy have emerged as two most important treatment strategies to treat cancer in clinical practice;however,to improve anticancer efficacy,combination chemotherapy still remains challenge.Dichloroacetate(DCA)could produce significant cytotoxic effects in certain tumor cells through its distinct mechanism.Radiation therapy with fast neutrons(FNT)has high relative biolgical effectiveness compared to other radiotherapeutics.Herein,we reported the combination chemotherapy with FNT for effective tumor growth inhibition with the assistance of a multilayered nanofiber loading DCA and DCA derivatives.We first synthesized a biodegradable polylysine to condense DCA with negative charge,or to conjugate DCA by condensing synthesis,to obtain Ion-DCA and Co-DCA,respectively.DCA,Ion-DCA or Co-DCA was then loaded into fibers to form multilayer drug-loaded mats.Upon adhesion on the surface of subcutaneous and orthotopic liver tumors,the multilayer drug-loaded mats realized a controllable release of DCA,which reversed the Warburg effect and inhibited cancer cell proliferation.Meantime,irradiation of fast neutrons could seriously damage DNA structure.Combination of the controllable release of DCA and FNT resulted in synergistic cell apoptosis in vitro,and the tumor inhibition in vivo.This study thus provides a new approach to integrate chemotherapy and FNT with the assistance of biocompatible nanofiber for synergistic tumor therapy.

Two sides to colon cancer: mice mimic human anatomical region disparity in colon cancer development and progression

Aim:Strong evidence reveals important differences between cancers in the proximal vs.distal colon.Animal models of metastatic colon cancer are available but with varying degrees of reproducibility and several important limitations.We explored whether there were regional differences in the location of murine colon cancers and assessed the utility of murine models to explore the biological basis for such differences.Methods:We re-analyzed data from our previous studies to assess the regional distribution of murine colon cancer.In survival surgery experiments,we injected HT-29 human colon cancer cells into the wall of the cecum or distal colon of Nu(NCr)-Foxn1 nu or NOD.Cg-Prkdc scid Il2rg Tim1Wji/SzJ mice and compared the development of primary tumors and metastases.Results:Within 7-17 weeks after intramural cecal injection of HT-29 cells,eight mice failed to develop solid primary tumors or metastases.In contrast,within four weeks after cell injection into the distal colon,13 mice developed metastases-12 mice developed subcutaneous metastases;of these,four developed liver metastases and one developed both liver and lung metastases.One mouse developed liver metastases only.Histological examination confirmed these lesions were adenocarcinomas.Conclusion:Our findings reveal the preferential growth of murine colon neoplasia and invasive human orthotopic xenografts in the distal mouse colon.The new approach of injecting cells into the distal colon wall results in a pattern of colon cancer development that closely mimics the progression of metastatic colon cancer in humans.This novel model of colon neoplasia has great potential for exploring anatomical differences in colon cancer and testing novel therapeutics.