Humanized Mouse Model Allows For Study Of Immune Response To Decellularized ECM Biomaterials

Jack Crawford on June 23, 2017

Humanized rodent models have been used extensively for studying autoimmune diseases, viral infections, xenogeneic transplantation and, more recently, allogenic stem cell transplantation; however, until now, researchers have not used these murine models for studies in the field of biomaterials. In their recent paper published in Biomaterials, Wang et al used a humanized mouse model to “assess the human immune response to decellularized extracellular matrix (ECM) biomaterials, specifically injectable hydrogels derived from porcine or human myocardium, which Biomaterials, Wang et al used a humanized mouse model to “assess the human immune response to decellularized extracellular matrix (ECM) biomaterials, specifically injectable hydrogels derived from porcine or human myocardium, which were initially developed to treat the heart post-myocardial infarction.”

This is important because the rodents typically used for biocompatibility testing provide limited representation of the human immune response because of differences in immune cell receptors, cytokine expression and response to various stimuli highlight how responses in rodents might not correlate with outcomes observed in humans. “Immune cells in the humanized mouse model, particularly T-helper cells, responded distinctly between the xenogeneic and allogeneic biomaterials,” according to Wang et al. “The allogeneic extracellular matrix derived hydrogels elicited significantly reduced total, human specific, and CD4 T-helper cell infiltration in humanized mice compared to xenogeneic extracellular matrix hydrogels, which was not recapitulated in wild type mice.”

Although this model certainly still has some limitations, the study was successful and gives high hopes to improving outcomes in the field of biomaterials, which continue to play an integral role in applications related to wound healing, hernia repair, skeletal muscle defect repair and hear attacks. As suggested by Wang et al, “decellularized ECM biomaterials are an attractive platform for biomaterial therapies since tissue derived from ECM can promote tissue remodeling by influencing cellular metabolism, proliferation, migration, maturation and differentiation” and these humanized mouse models will allow for further study of human immune cell responses to biomaterials in an in vivo environment.

Chimeric Humanized Mouse Models: Understanding Human And Mouse Cell Interactions

Jack Crawford on February 28, 2017

Humanized liver mouse models are increasingly being used in preclinical trials and have allowed for groundbreaking in-vivo research to evaluate everything from human-specific drug toxicity and efficacy to gene therapies. Unlike their transgenic mouse model counterparts, chimeric liver mouse models that include human hepatocytes and it is important for researchers to better understand the interactions between the implanted human cells and native mouse cells especially for drug metabolism studies.

In a recent study by Chow et al published in The Journal of Pharmacology and Experimental Therapeutics, it was shown that as a result of the species mismatch between human and mouse cells certain deficiencies are increasingly common, including dysregulation of hepatocyte proliferation and bile acid homeostasis in hFRGN livers that led to hepatotoxicity, gallbladder distension, liver deformity and other extrahepatic changes. “Although the nuclear receptors in human and other species share common targets, species difference in nuclear receptor activation exists”, and Chow et al suggest that additional research may be necessary to fully understand the inter-organ communication between human and mouse organs in h-chimeric mice.

Specifically, the miscommunication between human hepatocytes and murine stellate cells (which typically signal to hepatocytes to stop proliferating) is an important consideration. When this occurs, intracellular spaces are frequently reduced and cholangiocyte growth is inhibited, which can result in reduced bile flow as well as increased bile acid accumulation and toxicity.
Although we do not believe that any of these factors are reason enough to discontinue the use of humanized mouse or rat models for preclinical research, Chow et al do point out the need for increased awareness and the importance of addressing these deficiencies when reporting data in human drug metabolism studies.

Transposon Mutagenesis Helps Identify Genes Mediating Drug Resistance In The Treatment Of CLL

Jack Crawford on February 28, 2017

First time treatment of Chronic Lymphocratic Leukemia (CLL) generally requires a chemotherapy regimen that includes fludarabine. And although this potent drug combination has an astounding overall response rate of more than 90%, the unfortunate reality is that most patience will eventually relapse. And even more concerning is that in addition to the relatively small percentage of patients who are inherently resistant to fludarabine treatments from the start, with each subsequent use of this chemotherapy cocktail, data shows increased patient populations with acquired resistance to fludarabine-based chemotherapy, which ultimately presents a significant challenge for long-term disease control.

In order to better understand which specific genes and genetic pathways are mediating fludarabine resistance, Pandzic et al performed a “piggyBac transposon mutagenesis screen in a human CLL cell line” and their findings were recently reported in Clinical Cancer Research. In this study, a CLL cell line was subjected to random mutagenesis through integration of piggyBac transposons into genomic DNA. The cells were screened for resistance to fludarabine and the insertion sites of the piggyBac transposons identified through the use of Splinkerette PCR (spPCR). This screen not only revealed known resistance mediator genes, such as DCK (deoxycytidine kinase), but it also identified three novel genes, including BMP2K, which was shown to modulate response to fludarabine, although it had previously not been linked to CLL patients with fludarabine resistance.

This study demonstrated that piggyBac transposon mutagenesis screens have the ability to help lead the way when it comes to identifying genes that mediate patient sensitivity to specific drug therapies, including, but not limited to fludarabine-based chemotherapy. The piggyBac transposon is one of Hera BioLabs core technologies and we can assist with establishing various genome engineering and mutagenesis screens utilizing piggyBac in combination with CRISPR/Cas9.

Orthotopic vs. Subcutaneous Xenograft Models of Human Cancer

Jack Crawford on December 21, 2016

orthotopic xenografts and orthotopic PDXs for GBM in mice and rats Orthotopic vs. Subcutaneous Xenograft Models of Human Cancer

Subcutaneously implanted xenografts or patient-derived xenografts (PDXs) are a commonly used tool in the study of cancer.  Orthotopic xenografts are defined as the implantation of cancer cells into the same organ or tissue from which the cancer originated in the human, while subcutaneous xenografts are the implantation of cancer cells under the skin of an immunodeficient mouse or SRG rat.  Oncology researchers have differing opinions on which type of model to choose between when comparing orthotopic vs xenografts that are implanted subcutaneously (subq).

Why researchers choose to use a subcutaneous xenografts model:

  1. Ease-of-use: Subcutaneous xenografts are easier to implant, and monitor compared to orthotopic xenografts, which may require specialized surgical techniques. Subq implantation is an easy to learn technique and is not considered an invasive surgery.  Once tumor growth is observed, tumor volumes are easily measured by hand with a caliper enabling efficient tracking of tumor growth.
  2. Readily available models:  Since tumor measurements can be taken by calipers, the xenograft cell lines don’t need to be modified to express fluorescent or bioluminescent genes for assessment of tumor growth and there is a large number of already established models with historic data or publications for comparison when using a subcutaneous model.
  3. Cost considerations: Because orthotopic xenografts are more technically challenging to perform and require in vivo imaging to monitor tumor growth, researchers choose to not pursue the more resource intensive approach when preparing a cohort of animals for drug efficacy studies.

The advantages of choosing an orthotopic xenograft or PDX instead of using a subcutaneous model:

  1.  The tumor microenvironment of the xenograft or PDX implanted into the organ or tissue of origin may play a role in tumor growth and response to therapy. Orthotopic xenografts are thought to better mimic the native tumor microenvironment, including stromal cell components.
  2. Tumor growth rate: The rate of tumor growth may differ between orthotopic and subcutaneous xenografts due to differences in the tumor microenvironment and local nutrient supply. This may be particularly important when establishing PDXs which don’t have a high success rate implanted in the subcutaneous space.
  3. Tumor metastasis: Orthotopic xenografts may more accurately replicate the metastatic behavior of human tumors compared to subcutaneous xenografts.For example, orthotopic xenograft models more closely mimic the metastases observed in human prostate cancer patients, according to a 2016 study published in the Journal of Cellular Biochemistry by Zhang, et al.The results of this study clearly show “very different tumor behavior at the orthotopic and subcutaneous sites of human prostate cancer PC-3 in athymic nude mice,” according to Zhang, et al. “By day-2 after tumor implantation, the orthotopic tumor is already highly vascularized and the cancer cells have begun to migrate out of the tumor. In contrast, the subcutaneous tumor only begins to be vascularized by day-3 and cells to not migrate from the tumor.” Additionally, angiogenesis is much more extensive in the orthotopic tumor compared to the subcutaneous tumor over a two week period.Specifically, the orthotopic PC-3-GFP tumor is observed to grow very rapidly and presents distinct metastases in the lymph nodes by day-3 and evidence of metastases in the abdominal cavity by day-7. Compare this to the PDX model which, after a full 14 days, showed no evidence of invasion or metastasis associated with the subcutaneous tumor, even after the lymph nodes and abdominal cavities of the mouse was extensively explored.Using orthotopically-implanted PC-3-GFP cells, Zhang et al were also able to observe metastatic cells that migrated from the primary tumor to various organ systems, thereby demonstrating that “PC-3 has multiple metastatic routes similar to hormone-independent advanced-stage prostate cancer in the clinic.” As researchers continue to better understand the process by which metastases occur in PC-3 and other tumor types using orthotopic xenografts, the possibility for translational success in improved.

Overall, the choice between orthotopic and subcutaneous xenografts depends on the research question and the specific characteristics of the cancer model being studied. Both methods have advantages and disadvantages, and researchers should carefully consider the relevant factors before making a decision.

The SRG Rat for Orthotopic vs. Subcutaneous Xenografts

Whether you are considering subcutaneous or orthotopic approach to conducting your xenograft or PDX study, the SRG rat can provide certain advantages to both approaches.  First, the larger size of the SRG rat makes orthotopic xenograft implantations easier and less prone to error.  For example, sub renal/kidney capsule implants or colorectal both considered difficult surgeries, but with a larger rodent host there is more tolerance for successful implantation into the precise target tissue.  Secondly, the SRG rat shows a more human-like tumor microenvironment, even in the subcutaneous space, providing a more translational model for drug efficacy studies, and better tumor engraftment and growth kinetics regardless of site of tumor implantation.

The SRG rat is particularly applicable to the study of glioblastoma or other brain cancers. With these cancer types orthotopic xenografts are essential because having a intact blood-brain barrier is requirement to fully understand drug efficacy.  Surgical implantations can be very precise into various rat brain locations (including brain stem) with stereotactic injection equipment and the SRG rat tolerates a high tumor burden allowing for a longer window of study compared to mouse models.

You can learn more about how researchers are using the SRG rat HERE.

 

HIV-1 Gene Therapies Considered: RNAi Versus CRISPR-Cas

Jack Crawford on December 21, 2016

Though human immunodeficiency virus type 1 (HIV-1) can now be effectively treated to prevent disease progression through the use of potent antiviral drugs, decades after this disease’s discovery, there still is no cure for this precursor to acquired immune deficiency syndrome, also known as AIDS. In a recent article published in the journal Biochemical Society Transactions by Herrera-Carrillo and Berkhout from the Center for Infection and Immunity Amsterdam (CINIMA), new possible gene therapy approaches, including the use of RNAi and CRISPR-cas, are considered and discussed.

Though the end goal of each of these anti-HIV therapeutic options is the same – to stop the replication of HIV-1 – the two proposed mechanisms vary greatly in their biological origin: RNAi acts through suppressing mRNA and CRISPR targets the DNA. And although both RNAi and CRISPR-cas mechanisms both offer some significant hope for future clinical applications, neither is without risk and both may induce unwanted side effects at the cellular level. RNAi, for example, may cause “saturation or off-targeting of unrelated mRNAs” and CRISPR-cas can cause “permanent mutagenic effects” via cleavage of off-target DNA sequences, according to Herrera-Carrillo, et al.

The biggest problem with both RNAi and CRISPR-cas gene therapies is that one cannot reliably predict these adverse events, indicating that the safety and efficacy of each of these new gene therapies should be tested and verified in appropriate in vivo models. Herrera-Carrillo, et al tested the combinatorial RNAi therapy using a humanized immune system mouse model and a “single RNA-based anti-HIV gene therapy has moved into clinical trials” as a result. The CRISPR-cas system, however, still requires additional safety tests to more fully understand the “sustained expression of this foreign endonuclease in human cells, which may possibly lead to off-target cleavage events.”

The field of gene therapy has recently made great strides in the advancement of human applications. As additional in vivo testing in humanized rodent models continues, it is likely that researchers will develop increasingly efficient and safer therapeutic strategies designed to suppress the replication of HIV-1.

Advancements In Stem Cell-Based Therapies For Malignant Brain Tumors

Jack Crawford on October 26, 2016

Despite many great developments in the overall treatment of cancers, glioblastoma (GBM) patients still have a pretty dire prognosis, with an estimated 5% five year survival rate and a median survival of approximately 15 months from diagnosis. One of the biggest challenges associated with the treatment of malignant brain tumors, including GBM, is how to effectively deliver drugs to the site without causing serious adverse side effects – especially since any treatment therapy needs to cross the blood-brain barrier without losing significant potency, be able to diffuse throughout the brain and specifically target cancer cells rather than indiscriminately impacting all cells, including healthy cells.

Researchers have known for some time now that mesenchymal stem cells (MSCs), particularly human adipose-derived mesenchymal stem cells (hAMSCs), have great potential as brain tumor-targeting carriers of drug therapies. Until recently, it was widely accepted that the most effective way of engineering hAMSCs for drug delivery was through the use of viral vectors; unfortunately, large scale preclinical trials of such virus delivered gene therapy have raised significant questions about the risk of immunotoxicity as well as the potential activation of latent viruses, inflammatory responses or systemic autoimmunity.

A team from Johns Hopkins University School of Medicine, however, has recently published a study in Biomaterials that demonstrated the successful use of non-viral nanobiotechnology to deliver safe and effective gene and cell therapies in the treatment of GBM. By using synthetic nanoparticles (NPs), they were able to engineer hAMSCs to produce BMP4, a growth factor known to decrease tumor growth, which allowed for successful migration past various biological barriers and ultimately delivered a “therapeutic payload”. Using a cancer xenograft rat model, Mangravati et all were able to provide in vivo evidence that “NP-engineered hAMSCs administered locally and systemically in a rodent glioma model retain their intrinsic tumor-homing efficiency by migrating towards the brain and penetrating the tumor, and hAMSCs engineered to secrete BMP4 significantly increased survival” in brain tumor initiating cell-bearing rats without producing the debilitating side effects associated with viral vectors. This gives great hope to the future of drug and stem-cell based therapies for a wide array of human diseases, including, but not limited to, malignant brain cancer such as glioblastoma.