Immune humanization of immunodeficient mice using diagnostic bone marrow aspirates from carcinoma patients.

Melanie Werner-Klein,Roman Ganzer,Hans-Stefan Hofmann,Daniela N Männel,Ulrich Nitsche,Birgit Seelbach-Göbel,Andreas Blana,Stefan Buchholz,Brigitte Rack,Christian Werno,Christoph A Klein,Katharina Schneider,Judith Proske

doi:10.1371/journal.pone.0097860

Abstract

Tumor xenografts in immunodeficient mice, while routinely used in cancer research, preclude studying interactions of immune and cancer cells or, if humanized by allogeneic immune cells, are of limited use for tumor-immunological questions. Here, we explore a novel way to generate cancer models with an autologous humanized immune system. We demonstrate that hematopoietic stem and progenitor cells (HSPCs) from bone marrow aspirates of non-metastasized carcinoma patients, which are taken at specialized centers for diagnostic purposes, can be used to generate a human immune system in NOD-scid IL2rγ(null) (NSG) and HLA-I expressing NSG mice (NSG-HLA-A2/HHD) comprising both, lymphoid and myeloid cell lineages. Using NSG-HLA-A2/HHD mice, we show that responsive and self-tolerant human T cells develop and human antigen presenting cells can activate human T cells. As critical factors we identified the low potential of bone marrow HSPCs to engraft, generally low HSPC numbers in patient-derived bone marrow samples, cryopreservation and routes of cell administration. We provide here an optimized protocol that uses a minimum number of HSPCs, preselects high-quality bone marrow samples defined by the number of initially isolated leukocytes and intra-femoral or intra-venous injection. In conclusion, the use of diagnostic bone marrow aspirates from non-metastasized carcinoma patients for the immunological humanization of immunodeficient mice is feasible and opens the chance for individualized analyses of anti-tumoral T cell responses.

Highlights

The immunobiology of solid tumors is mostly studied in small animal models, primarily in mice
To achieve long-term reconstitution of NSG mice with human immune cells, an intrahepatic injection of CD34+ hematopoietic stem and progenitor cells (HSPCs) into newborn NSG mice is currently considered to result in best engraftment [13]
As the arrival of diagnostic bone marrow aspirates cannot be scheduled and rarely matches with the availability of newborn mice, we tested whether intra-femoral injections of BM-derived CD34+ HSPC into adult NSG mice gives a comparable reconstitution aspirates were isolated by density centrifugation and frozen in liquid nitrogen

Summary

Introduction

The immunobiology of solid tumors is mostly studied in small animal models, primarily in mice. These studies are mainly based on tissue- or cell-specific expression of oncogenes, chemicalinduced carcinogenesis or spontaneously arising tumors in immunocompetent mice [1]. Immunodeficient mice are used to establish xenografts of human tumors, but these models suffer from either the complete absence of human immune cells or poor engraftment of certain hematopoietic lineages after hematopoietic stem and progenitor cell (HSPC) transplantation. Simultaneous transplantation of tumor cell lines with cord-blood derived HSPCs [7] demonstrated that both cell types could successfully co-engraft. Transplantation of syngeneic immune and cancer cells would be needed

Methods

Results

Conclusion