Dual use of hematopoietic and mesenchymal stem cells enhances engraftment and immune cell trafficking in an allogeneic humanized mouse model of head and neck cancer

Abstract

In this report, we describe in detail the evolving procedures to optimize humanized mouse cohort generation, including optimal conditioning, choice of lineage for engraftment, threshold for successful engraftment, HNSCC tumor implantation, and immune and stroma cell analyses. We developed a dual infusion protocol of human hematopoietic stem and progenitor cells (HSPCs) and mesenchymal stem cells (MSCs), leading to incremental human bone marrow engraftment, and exponential increase in mature peripheral human immune cells, and intratumor homing that includes a more complete lineage reconstitution. Additionally, we have identified practical rules to predict successful HSPC/MSC expansion, and a peripheral human cell threshold associated with bone marrow engraftment, both of which will optimize cohort generation and management. The tremendous advances in immune therapy in cancer have made the need for appropriate and standardized models more acute than ever, and therefore, we anticipate that this manuscript will have an immediate impact in cancer-related research. The need for more representative tools to investigate the human tumor microenvironment (TME) has led to the development of humanized mouse models. However, the difficulty of immune system engraftment and minimal human immune cell infiltration into implanted xenografts are major challenges. We have developed an improved method for generating mismatched humanized mice (mHM), using a dual infusion of human HSPCs and MSCs, isolated from cord blood and expanded in vitro. Engraftment with both HSPCs and MSCs produces mice with almost twice the percentage of human immune cells in their bone marrow, compared to mice engrafted with HSPCs alone, and yields 9- to 38-fold higher levels of mature peripheral human immune cells. We identified a peripheral mHM blood human B cell threshold that predicts an optimal degree of mouse bone marrow humanization. When head and neck squamous cell carcinoma (HNSCC) tumors are implanted on the flanks of HSPC-MSC engrafted mice, human T cells, B cells, and macrophages infiltrate the stroma of these tumors at 2- to 8-fold higher ratios. In dually HSPC-MSC engrafted mice we also more frequently observed additional types of immune cells, including regulatory T cells, cytotoxic T cells, and MDSCs. Higher humanization was associated with in vivo response to immune-directed therapy. The complex immune environment arising in tumors from dually HSPC-MSC engrafted mice better resembles that of the originating patient's tumor, suggesting an enhanced capability to accurately recapitulate a human TME.

The humanized mouse: Emerging translational potential

“…More recently, researchers have determined that, whatever their other effects, injected MSCs do not alter the tumor immune response in HM 21 . In accordance with much of this data, we have shown that the isolation, coculture, and coinjection of cord blood–derived MSCs together with HSCs can produce HM with not only a higher percentage of human bone marrow cells but also with a greater percentage of circulating T and B cells, as well as monocytes 51 …”

“…Although several groups have reported tumor regression after administration of these agents, in many cases it is uncertain how this response compares to that of a patient 26,75 . We have observed tumor regression in response to nivolumab treatment, but only in very well‐humanized mice, and only using a PDX taken from a patient who was not treated with these immune checkpoint inhibitors 51 . When these HM contained even marginally less human bone marrow, we could not see this treatment effect.…”

“…Conversely, T cells have been observed in HM almost a year after their engraftment, indicating that these cells can persist within this model for an extended period 10 . Futhermore, it has also been observed that engrafted MSCs within the HM can help increase their presence within the HM 51 …”

“…A subsequent study showed that the subcutaneous implantation of cord blood–derived MSCs (identified as mesenchymal stem/progenitor cells therein) in NSG mice effectively recruited HIF‐1α, leading to the upregulation of vascular endothelial growth factor and stimulation of neovascularization 67 . Additional studies have shown that changes in the TME are also driven by infiltrating immune cells 68 and that such cells are quite capable of infiltrating this compartment within the HM PDX, especially when the HM is generated using both HSCs and MSCs 10,51,59,69 . Finally, it has also been observed that breast tumor biopsies orthotopically implanted into HM models retained their ability to metastasize into the lungs 13 …”

Mouse Tumor-Bearing Models as Preclinical Study Platforms for Oral Squamous Cell Carcinoma

“…Therefore, the XactMice system accurately recapitulates the growth of the original tumor in vivo. The humanized mouse model of HNSCC was optimized in another study by Morton et al (163). After the dual infusion of human hematopoietic stem and progenitor cells (HSPCs) and mesenchymal stem cells (MSCs), the percentage of human immune cells in the bone marrow of established mice was almost twice that in mice implanted with HSPCs alone, and mature peripheral human immune cells were 9-38-fold more abundant.…”