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 - PubMed (original) (raw)

. 2018 Nov;57(11):1651-1663.

doi: 10.1002/mc.22887. Epub 2018 Sep 3.

Stephen B Keysar 1, Loni Perrenoud 1, Tugs-Saikhan Chimed 1, Julie Reisinger 1, Brian Jackson 1, Phuong N Le 1, Cera Nieto 1, Karina Gomez 1, Bettina Miller 1, Dexiang Gao 2, Hilary Somerset 3, Xiao-Jing Wang 3 4 5, Antonio Jimeno 1 4

Affiliations

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

John J Morton et al. Mol Carcinog. 2018 Nov.

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.

Keywords: cancer microenvironment; head and neck cancer; hematopoietic stem cell; humanized mouse model; patient-derived xenograft.

© 2018 Wiley Periodicals, Inc.

PubMed Disclaimer

Conflict of interest statement

Competing financial interests

The authors declare that they have no potential conflicts of interest.

Figures

Figure 1.

Figure 1.. In vitro amplification of HSPCs and MSCs; pre-engraftment mouse irradiation.

(a) Growth curves of HSPCs purified from responsive and non-responsive cords. Significant differences in proliferation can be seen after several days in culture (mid-expansion). (b) Flow cytometry depicting the amplification of HSPCs during in vitro expansion and, by day 13, the gradual loss of HSPCs due to increased immune cell differentiation. The total number of cells in the sample is recorded above each graph, along with the percentage of those cells (in green, corresponding to the green box superimposed on the graph) that are HSPCs. (c) Chart comparing potential and actual sizes of the mHM cohorts after HSPC expansion, based on an injection of 400,000 HSPCs per mouse. Cohorts HM057 and HM069 were generated using 300,000 cells/mouse, so their actual size is greater than their potential size. (d) HSPC loss due to freezing and subsequent re-expansion after removal of the cells from cryopreservation. Overall averages for all subsequently expanded cryopreserved HSPCs (red) and for the HM084 HSPCs (blue) are shown. For HM084, the cell numbers at the blue and green points on this graph are represented by cytometry, which shows pre-and post-freeze HM084 HSPCs in culture. (e) Picture of HSPCs (non-adherent circular cells) and MSCs (adherent spindle-like cells) in culture. Magnification is 10x and scale bar = 200μm. (f) Cytometry illustrating the relative abundance of HSPCs and MSCs at the end of in vitro expansion. (g) Cytometry indicates that many of the MSCs also express CD90 and CD105. (h) Relative expansion of HSPCs and MSCs from responsive cords in culture. (i) Comparison of radiation-induced mortality. Irradiation greater than 1.5 Gy resulted in 2.8 times as many deaths

Figure 1.

Figure 1.. In vitro amplification of HSPCs and MSCs; pre-engraftment mouse irradiation.

(a) Growth curves of HSPCs purified from responsive and non-responsive cords. Significant differences in proliferation can be seen after several days in culture (mid-expansion). (b) Flow cytometry depicting the amplification of HSPCs during in vitro expansion and, by day 13, the gradual loss of HSPCs due to increased immune cell differentiation. The total number of cells in the sample is recorded above each graph, along with the percentage of those cells (in green, corresponding to the green box superimposed on the graph) that are HSPCs. (c) Chart comparing potential and actual sizes of the mHM cohorts after HSPC expansion, based on an injection of 400,000 HSPCs per mouse. Cohorts HM057 and HM069 were generated using 300,000 cells/mouse, so their actual size is greater than their potential size. (d) HSPC loss due to freezing and subsequent re-expansion after removal of the cells from cryopreservation. Overall averages for all subsequently expanded cryopreserved HSPCs (red) and for the HM084 HSPCs (blue) are shown. For HM084, the cell numbers at the blue and green points on this graph are represented by cytometry, which shows pre-and post-freeze HM084 HSPCs in culture. (e) Picture of HSPCs (non-adherent circular cells) and MSCs (adherent spindle-like cells) in culture. Magnification is 10x and scale bar = 200μm. (f) Cytometry illustrating the relative abundance of HSPCs and MSCs at the end of in vitro expansion. (g) Cytometry indicates that many of the MSCs also express CD90 and CD105. (h) Relative expansion of HSPCs and MSCs from responsive cords in culture. (i) Comparison of radiation-induced mortality. Irradiation greater than 1.5 Gy resulted in 2.8 times as many deaths

Figure 1.

Figure 1.. In vitro amplification of HSPCs and MSCs; pre-engraftment mouse irradiation.

(a) Growth curves of HSPCs purified from responsive and non-responsive cords. Significant differences in proliferation can be seen after several days in culture (mid-expansion). (b) Flow cytometry depicting the amplification of HSPCs during in vitro expansion and, by day 13, the gradual loss of HSPCs due to increased immune cell differentiation. The total number of cells in the sample is recorded above each graph, along with the percentage of those cells (in green, corresponding to the green box superimposed on the graph) that are HSPCs. (c) Chart comparing potential and actual sizes of the mHM cohorts after HSPC expansion, based on an injection of 400,000 HSPCs per mouse. Cohorts HM057 and HM069 were generated using 300,000 cells/mouse, so their actual size is greater than their potential size. (d) HSPC loss due to freezing and subsequent re-expansion after removal of the cells from cryopreservation. Overall averages for all subsequently expanded cryopreserved HSPCs (red) and for the HM084 HSPCs (blue) are shown. For HM084, the cell numbers at the blue and green points on this graph are represented by cytometry, which shows pre-and post-freeze HM084 HSPCs in culture. (e) Picture of HSPCs (non-adherent circular cells) and MSCs (adherent spindle-like cells) in culture. Magnification is 10x and scale bar = 200μm. (f) Cytometry illustrating the relative abundance of HSPCs and MSCs at the end of in vitro expansion. (g) Cytometry indicates that many of the MSCs also express CD90 and CD105. (h) Relative expansion of HSPCs and MSCs from responsive cords in culture. (i) Comparison of radiation-induced mortality. Irradiation greater than 1.5 Gy resulted in 2.8 times as many deaths

Figure 2.

Figure 2.. Evidence of humanization in mHM cohorts.

(a) Comparison of the average percentage of human CD45+ immune cells within the bone marrow of cohorts engrafted with HSPCs only (first six cohorts on the left, shaded in gray) and those engrafted with an HSPC- MSC mixture, along with the number of mice in each cohort. (b) Chart comparing the average CD45+ cell percentage (light green) and the average CD34-45+ HSPCs percentage (dark green) in mouse cohorts generated using only HSPCs and those created from HSPC-MSCs. *P=0.007. (c) Comparison of the percentage of human T cells, B cells, and monocytes found in the blood of mice engrafted with HSPCs and HSPC-MSCs. *P=<0.01, **P=0.01, ***P<0.01. Also compared are the percentages of total human CD45+ cells observed in these two mouse types. ****P<0.01. (d) Comparison of the percentage of human T cells, B cells, and monocytes found in the spleen of mice engrafted with HSPCs and MSPC-MSCs. *P<0.01, **P<0.01, ***P<0.01.

Figure 3.

Figure 3.. Defining the degree of humanization at which immune cells infiltrate into implanted tumors.

(a) Cytometry showing animals with a higher percentage of human immune cells in their bone marrow are also more likely to have human immune cells within their peripheral blood and infiltrating implanted tumors. (b) Panel 1: Graph showing the relationship between immune cells in the bone marrow (red line) and immune cell infiltration into tumors (circles), organized by mouse – ranked according to their percentage of human bone marrow – on the X axis. The arrow indicates the mouse identified as the changepoint between poorly- and well-humanized mice. Panel 2: Changepoint analysis showing the increase in the mean percentage of immune infiltration in mice whose bone marrow is composed of at least 20% human immune cells. The arrow indicates the changepoint mouse. (c) Plots depicting increased human B cell content in the peripheral blood of well-humanized mice (*P=0.01; left) and increased immune cells infiltration in tumors implanted on well-humanized mice (**P<0.01; right).

Figure 4.

Figure 4.. Characteristics of immune cell infiltration in implanted tumors.

(a) IHC comparison of the relative presence of T cells (top panel), B cells (middle panel), and macrophages (bottom panel) within the tumors of well humanized mice engrafted either with HSPCs only (CUHN013 from HM005, whose bone marrow is composed of 32.74% human cells) or a HSPC-MSCs (CUHN004 from HM034, 91.69% human bone marrow). Magnification is 20x; scale bar = 100μm. (b) Comparison of the percentage of human T cells, B cells, and monocytes found in the tumors implanted on all tumor-bearing cohorts of mice engrafted with HSPCs and HSPC-MSCs. *P<0.01, **P=0.03, ***P=0.01. (c) Cytometry comparing the relative abundance of uncharacterized human CD45+ immune cells present in tumors implanted on mice engrafted with HSPCs (HM005) and HSPC-MSCs (HM034). In the HM005 mouse, immune cells make up 0.38% (average of the values in the colored boxes) of all cells in the tumor, yet T cells, B cells, and macrophages account for only 16% of all cells. In the HM034 mouse, immune cells make up 1.64% of all cells, while T cells, B cells and macrophages account for 1.21% of all cells in the tumor. (d) IHC staining for T cells, B cells, macrophages, NK cells, Treg cells, cytotoxic T cells, and eMDSCs within patient tumors and tumors implanted on mice engrafted with HSPCs or HSPC-MSCs. Top series = CUHN013 tumor from patient, from HM004 mouse, and from HM045 mouse. Bottom series = CUHN022 tumor from patient, from HM010 mouse, and from HM069 mouse. Magnification is 20x; scale bar = 100μm. (e) IHC staining comparing M1 and M2 macrophage differentiation within patient tumors and xenografts. Magnification is 20x; scale bar = 100μm.

Figure 4.

Figure 4.. Characteristics of immune cell infiltration in implanted tumors.

(a) IHC comparison of the relative presence of T cells (top panel), B cells (middle panel), and macrophages (bottom panel) within the tumors of well humanized mice engrafted either with HSPCs only (CUHN013 from HM005, whose bone marrow is composed of 32.74% human cells) or a HSPC-MSCs (CUHN004 from HM034, 91.69% human bone marrow). Magnification is 20x; scale bar = 100μm. (b) Comparison of the percentage of human T cells, B cells, and monocytes found in the tumors implanted on all tumor-bearing cohorts of mice engrafted with HSPCs and HSPC-MSCs. *P<0.01, **P=0.03, ***P=0.01. (c) Cytometry comparing the relative abundance of uncharacterized human CD45+ immune cells present in tumors implanted on mice engrafted with HSPCs (HM005) and HSPC-MSCs (HM034). In the HM005 mouse, immune cells make up 0.38% (average of the values in the colored boxes) of all cells in the tumor, yet T cells, B cells, and macrophages account for only 16% of all cells. In the HM034 mouse, immune cells make up 1.64% of all cells, while T cells, B cells and macrophages account for 1.21% of all cells in the tumor. (d) IHC staining for T cells, B cells, macrophages, NK cells, Treg cells, cytotoxic T cells, and eMDSCs within patient tumors and tumors implanted on mice engrafted with HSPCs or HSPC-MSCs. Top series = CUHN013 tumor from patient, from HM004 mouse, and from HM045 mouse. Bottom series = CUHN022 tumor from patient, from HM010 mouse, and from HM069 mouse. Magnification is 20x; scale bar = 100μm. (e) IHC staining comparing M1 and M2 macrophage differentiation within patient tumors and xenografts. Magnification is 20x; scale bar = 100μm.

Figure 4.

Figure 4.. Characteristics of immune cell infiltration in implanted tumors.

(a) IHC comparison of the relative presence of T cells (top panel), B cells (middle panel), and macrophages (bottom panel) within the tumors of well humanized mice engrafted either with HSPCs only (CUHN013 from HM005, whose bone marrow is composed of 32.74% human cells) or a HSPC-MSCs (CUHN004 from HM034, 91.69% human bone marrow). Magnification is 20x; scale bar = 100μm. (b) Comparison of the percentage of human T cells, B cells, and monocytes found in the tumors implanted on all tumor-bearing cohorts of mice engrafted with HSPCs and HSPC-MSCs. *P<0.01, **P=0.03, ***P=0.01. (c) Cytometry comparing the relative abundance of uncharacterized human CD45+ immune cells present in tumors implanted on mice engrafted with HSPCs (HM005) and HSPC-MSCs (HM034). In the HM005 mouse, immune cells make up 0.38% (average of the values in the colored boxes) of all cells in the tumor, yet T cells, B cells, and macrophages account for only 16% of all cells. In the HM034 mouse, immune cells make up 1.64% of all cells, while T cells, B cells and macrophages account for 1.21% of all cells in the tumor. (d) IHC staining for T cells, B cells, macrophages, NK cells, Treg cells, cytotoxic T cells, and eMDSCs within patient tumors and tumors implanted on mice engrafted with HSPCs or HSPC-MSCs. Top series = CUHN013 tumor from patient, from HM004 mouse, and from HM045 mouse. Bottom series = CUHN022 tumor from patient, from HM010 mouse, and from HM069 mouse. Magnification is 20x; scale bar = 100μm. (e) IHC staining comparing M1 and M2 macrophage differentiation within patient tumors and xenografts. Magnification is 20x; scale bar = 100μm.

Figure 4.

Figure 4.. Characteristics of immune cell infiltration in implanted tumors.

(a) IHC comparison of the relative presence of T cells (top panel), B cells (middle panel), and macrophages (bottom panel) within the tumors of well humanized mice engrafted either with HSPCs only (CUHN013 from HM005, whose bone marrow is composed of 32.74% human cells) or a HSPC-MSCs (CUHN004 from HM034, 91.69% human bone marrow). Magnification is 20x; scale bar = 100μm. (b) Comparison of the percentage of human T cells, B cells, and monocytes found in the tumors implanted on all tumor-bearing cohorts of mice engrafted with HSPCs and HSPC-MSCs. *P<0.01, **P=0.03, ***P=0.01. (c) Cytometry comparing the relative abundance of uncharacterized human CD45+ immune cells present in tumors implanted on mice engrafted with HSPCs (HM005) and HSPC-MSCs (HM034). In the HM005 mouse, immune cells make up 0.38% (average of the values in the colored boxes) of all cells in the tumor, yet T cells, B cells, and macrophages account for only 16% of all cells. In the HM034 mouse, immune cells make up 1.64% of all cells, while T cells, B cells and macrophages account for 1.21% of all cells in the tumor. (d) IHC staining for T cells, B cells, macrophages, NK cells, Treg cells, cytotoxic T cells, and eMDSCs within patient tumors and tumors implanted on mice engrafted with HSPCs or HSPC-MSCs. Top series = CUHN013 tumor from patient, from HM004 mouse, and from HM045 mouse. Bottom series = CUHN022 tumor from patient, from HM010 mouse, and from HM069 mouse. Magnification is 20x; scale bar = 100μm. (e) IHC staining comparing M1 and M2 macrophage differentiation within patient tumors and xenografts. Magnification is 20x; scale bar = 100μm.

Figure 5.

Figure 5.. Mouse humanization and resultant T cell infiltration and activity within tumors dictate nivolumab response.

(a) Human immune cells, including T cells (identified by dual redbrown staining) are much more abundant in both control and nivolumab-treated CUHN004 tumors implanted on very well-humanized mice (HM034, average 82.4% human bone marrow) than in comparable tumors on a more moderately humanized cohort (HM044, average 31.5% human bone marrow.) (b) T-cell mediated cell killing (identified by granzyme B, stained brown) in the vicinity of CD3+ T cells (stained red) is a measure of the activity of tumor-infiltrating T cells in these cohorts before and after nivolumab treatment. The T/C of 0.70 in the HM034 cohort suggests that, when T cells are present in great enough numbers within a tumor, nivolumab can enhance their ability to destroy tumor cells.

Similar articles

Cited by

References

    1. Ferris RL, Blumenschein G Jr., Fayette J, Guigay J, Colevas AD, Licitra L, et al. Nivolumab for Recurrent Squamous-Cell Carcinoma of the Head and Neck. The New England journal of medicine 2016;375(19):1856–67 doi 10.1056/NEJMoa1602252. - DOI - PMC - PubMed
    1. Gillet JP, Varma S, Gottesman MM. The clinical relevance of cancer cell lines. J Natl Cancer Inst 2013;105(7):452–8 doi 10.1093/jnci/djt007. - DOI - PMC - PubMed
    1. Ben-David U, Ha G, Tseng YY, Greenwald NF, Oh C, Shih J, et al. Patient-derived xenografts undergo mouse-specific tumor evolution. Nat Genet 2017;49(11):1567–75 doi 10.1038/ng.3967. - DOI - PMC - PubMed
    1. Day CP, Merlino G, Van Dyke T. Preclinical mouse cancer models: a maze of opportunities and challenges. Cell 2015;163(1):39–53 doi 10.1016/j.cell.2015.08.068. - DOI - PMC - PubMed
    1. Shultz LD, Lyons BL, Burzenski LM, Gott B, Chen X, Chaleff S, et al. Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells. Journal of Immunology (Baltimore, Md: 1950) 2005;174:6477–89. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources