Ex vivo analysis of mouse models

Given the similarity to the human genome, the versatility and the ease of use of mouse models makes them the most used animal models in drug discovery. They are of great importance for lead optimization studies and preclinical toxicity assessments of novel candidate therapeutics for complex diseases, such as cancer and autoimmune diseases. Whether working with syngeneic, xenograft, genetically modified, or humanized mouse models, Miltenyi Biotec offers efficient and gentle solutions to derive and characterize viable target cells

Patient-derived xenografts are a useful mouse model for drug development studies given their faithful representation of the patient’s original tumor immunohistochemically, genetically, and in terms of response to common therapeutics. However, as a consequence of vascularization of tumor tissue, these models provide tumor samples which are contaminated by a variable amount of murine stromal cells. This leads to biased molecular analysis, like microarray-based expression profiling and next-generation sequencing (NGS). In order to overcome these limitations, we have developed a fast and easy method for the effective depletion of mouse cells by combining optimized tumor dissociation with automated magnetic cell sorting. 

Read counts of bulk tumor and isolated human tumor cells

In this study by Agorku et al., tumor dissociation and magnetic cell separation led to an increase in read counts of 33% for the samples depleted of mouse cells, indicating improved sample quality. An average of 12% of reads derived from bulk samples was attributed to mouse cells and this amount could be reduced to 0.3% by prior depletion of mouse cells, thereby reducing the number of reads erroneously mapped to human genome and improving the sensitivity of SNP detection on human tumor xenografts.

Scientific poster
Next-generation sequencing of human tumor xenografts is significantly improved by prior depletion of mouse cells

David Agorku, Stefan Tomiuk, Kerstin Klingner, Stefan Wild, Silvia Rüberg, Lisa Zatrieb, Andreas Bosio, Julia Schueler, and Olaf Hardt

Techniques for the collection of CD45+ tumor-infiltrating lymphocytes (TILs) frequently can have major drawbacks. These include harsh tumor dissociations, which jeopardize cell viability and key surface epitopes, as well as density gradient-based cell isolation, which results in nonspecific cell enrichment and requires time-consuming protocols. Most magnetic cell separation options are not optimized for tumor material and thereby provide only low TIL purities. At Miltenyi Biotec, we have developed optimized dissociation of tumor samples and automated immunomagnetic enrichment of highly pure various TIL-subpopulations while preserving cell viability and integrity.

Isolation of TILs from human xenograft tumors using CD45 (TIL) MicroBeads

In this study by Gao et al., from Pfizer Inc., the combined mechanical and enzymatic dissocation of tumor samples resulted in single-cell suspensions that were appropriate for subsequent TIL isolation based on the CD45 epitope. After the TILs were isolated, an increase of CD45+ TIL frequencies could be seen from 29.6% and 29.3% (Tumor 1 and 2) to 88.6% and 91.9%, respectively, proving to be a reliable method to isolate highly pure TILs.

Application note
Effective enrichment of human tumor-infiltrating leukocytes from xenograft tumors

Ying Gao, Oncology R&D, Pfizer Inc., Pearl River, NY, USA

Syngeneic mouse tumor models represent the gold standard to analyze effects of immunotherapy and often rely on the characterization of TILs and their phenotype. However, the amount and composition of TILs is highly variable and often very low, complicating the flow cytometric analysis of individual subpopulations. To make things more difficult, traditional, hybridoma-derived antibody clones show a tendency of nonspecific binding to immune cells via Fcγ receptors (FcγR). This can lead to an inaccurate and biased estimation of TIL populations. Furthermore, they increase staining time and hinder scale up or automation of multi-sample analyses. We have developed REAfinity™ Recombinant Antibody clones characterized by a specifically mutated human IgG1 Fc region that abolishes their binding to FcγRs and therefore enables more reliable and reproducible flow cytometric analyses of immune cells.

Background staining of different immune cell subsets in the tumor microenvironment when using hybridoma-derived antibodies and REAfinity Antibodies

In this study, we show the difference when using REAfinity Recombinant Antibody clones versus hybridoma antibody clones for the analysis of TIL populations. Hybridoma-derived antibodies lead to a gross overestimation of TIL frequency of immune cell subpopulations present in the tumor. This was likely due to nonspecific binding to FcγRs on immune cells. The REAfinity Recombinant Antibodies provided a more exact analysis of the respective cell populations, even in the absence of FcR blocking.

Application note
Background-free analysis of mouse TILs 

Read more about the study describing the effective use REAfinity™ Recombinant Antibody clones for the analysis of different TIL populations.

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