We would like to thank both the Animal Research Facility (OLAR) for their care of our mice, and the Flow Cytometry Shared Resource supported by the Cancer Center Support Grant (P30CA046934) at our institute for their immense help in all our work. We also acknowledge both Gail Eckhardt and Anna Capasso for our inaugural collaborations studying immunotherapies to human PDXs in our HIS-BRGS model.&#160;This study was supported in part by the National Institutes of Health P30CA06934 Cancer Center Support Grant with use of the PHISM (Pre-clinical Human Immune System Mouse Models) Shared Resource, RRID: SCR_021990 and Flow Cytometry Shared Resource, RRID: SCR_022035.&#160;This research was supported in part by the NIAID of the National Institutes of Health under Contract Number 75N93020C00058.

<ol>
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Over the past 6 years, using our expertise in both immunology and humanized mice, our research team has developed a much needed preclinical model to test immunotherapies on a variety of human tumors3,7,30,31. This protocol emphasizes the consideration of the variability of the model, with special attention to the immunotherapy-centric human T cell populations. In this protocol, the generation of HIS mice is described, as well as the immune analysis of both their lymph organs and the tumor. Flow cytometry protocols developed for traditional compensation-based cytometers and spectral cytometers have also been included. The ability to interrogate changes in the immune system in large tumor samples, as well as in the periphery, represents a significant advantage to this preclinical model. An additional advantage to this system is that multiple mice, harboring the same unique human tumor, can be allocated into separate cohorts and treated with different combinations of immunotherapy drugs, essentially performing a &#34;mini&#34; drug trial. Combined, these two factors enable investigation into the mechanisms of action and resistance of proposed (combination) immunotherapies. The model has the ability to test drug timing and dosing on a wide variety of human tumors and a multitude of immune-targeted treatments, including targeted therapies, biologics, chemotherapies, radiation, and even cell-based therapies.
Due to the inherent variability in the HIS mice chimerism, a higher number of mice per treatment group is required than a typical syngeneic tumor model. Using these protocols, cohorts of five to seven HIS-BRGS mice per treatment arm are sufficient to obtain statistical differences in immune parameters. This variability in chimerism represents a challenge in this model and must be considered. An experimental example demonstrating both tumor growth and immune response upon a combination treatment to a CRC PDX has been shown here, as well as an example in which the same treatment to a different CRC PDX did not show a similar strong response. This difference in experimental outcome could be due to the different PDX (same patient but metastatic vs. primary) and/or the chimerism. In the second experiment, there were very few T cells in the spleen of the majority of mice in the treatment group, as well as very small LNs; several labs have shown that the largest batch-to-batch variability among HIS-models are the T cell frequencies20,32,33. We have also shown that human engraftment in the LNs correlates highly with the T cell chimerism32. Across more than 40 experiments, we have noted a requirement of 20% T cells in the spleen for adequate chimerism to detect treatment-related immune changes3. Upon sacrifice, it is important to ensure significant T cell reconstitution, which we define as notable LN development (&#62;1.5 x 106 hCD45 cells) and &#62;20% T cells (of hCD45+ cells) in the spleen. HIS mice that do not meet this requirement are removed from the data set. Chimerism in the blood prior to tumor implant helps predict this parameter; however, there are differences in T cell development in individual mice over time that are best considered at the end of the study. Fortunately, in this model, approximately 95% of HIS-BRGS-mice cohorts develop sufficient T cell chimerism for immunotherapy studies. It should be emphasized that the data analysis at the end of the study should always include interrogation of the chimerism in the peripheral lymph organs and remove HIS mice that do not meet the human and T cell chimerism. In experiments with less than four HIS mice per cohort following this adjustment, the results should be validated with a different HIS-BRGS cohort derived from a distinct CB.
Although our group has focused on the BRGS mouse model recipient, there are numerous models available worldwide. For instance, the NSG (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ)recipient is the most widely characterized model and is similar to the NOG (NOD.Cg-PrkdcscidIl2rgtm1Sug/Jic) and even the NRG (NOD-Rag1tm1MomIl2rgtm1Wjl/SzJ) model, which uses Rag mutations for genetic ablation of mouse T and B cells instead of the SCID mutation20. These models are all on the NOD background strain and thus have the SIRPaNOD allele, which is important in avoiding phagocytosis of human cells by host macrophages13. Reports suggest similar chimerism in these &#34;basic&#34; humanized mouse models as in the BRGS model, with mostly human B cells in the first few months followed by engraftment with human T cells32,34,35. In these models, the NK and myeloid cells are underrepresented relative to a human36,37. There are several iterations of &#34;next gen&#34; humanized mouse recipients that can improve lineage-specific development through the introduction of human-specific cytokines. For example, NSG-SGM3 (NOD.Cg-PrkdcscidIl2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ) mice have human IL-3, GM-CSF, and SCF cytokines and improved human myelopoiesis37, or human HLA transgenes for selection on a human HLA (e.g., NSG-HLA-A2 or BRGS-HLA-A2/DR238,39). Extensive reviews on this topic20,21 are recommended to readers. HIS mouse models should be validated in each laboratory with their own source of human stem cells. This validation should include quantifying the human chimerism, including human B, T, NK, and myeloid cells, in the blood over time and in the LNs, spleen, bone marrow, and thymus between 16-20 weeks and again between 24-28 weeks. Special attention should be given to multi-lineage engraftment, including T cell chimerism. Additionally, the growth rates of tumors should be tested in the HIS mice relative to non-humanized mice of the same strain. It is plausible that significant improvements in HIS mouse models could result in rejection of the tumor. As a resource-savings attempt, these growth curves are often done simultaneously with the immune response data analyses. Commercially available HIS mice should include extensive chimerism data from the blood as well as references to chimerism in other organs from other cohorts.
The experimental protocols for analyzing tumor growth and immune responses in HIS mice to human CDXs or PDXs require standard laboratory methodologies and immunological expertise. The generation of HIS mice, including the isolation of the stem cells, injection into immunodeficient mice, and testing of human chimerism in the blood, are all relatively straightforward lab procedures. The logistical issues surrounding this procedure, including breeding highly immunodeficient mouse strains, acquiring a reliable source of human HSCs, etc., are the most cumbersome component of the protocol. Procedurally, more attention must be given to experimental design, including the timing of tumor implantation (typically 19-21 weeks when enough T cells are present), drug dosing, and end-of-study timing. Acquiring tumor measurement data is another routine procedure. However, since the immune data is often more informative than the tumor growth data in this system, flow cytometric analyses of the immune responses can provide pertinent data. The staining of cells for flow cytometry is another straightforward procedure, as is learning how to run a flow cytometer. However, the analysis and interpretation of this data requires unique expertise. While this manuscript serves as a backbone to carry out immuno-oncology studies in HIS mice, each protocol step should be optimized within a new lab. From experience, maintaining the health of the mice, acquiring adequate HSCs from CB units, and timing the tumor injections for sufficient T cells require the most attention and troubleshooting.
The HIS-BRGS mice exhibit extreme immune suppression similar to that observed in many human tumors. This immune suppression is notable, as high expression levels of PD-1 and TIGIT, yet less TIM-3, are observed on the T cells in the peripheral immune organs of the HIS-BRGS mice3,7. These T cells also show markers of immune activation, including high expression of HLA-DR and low expression of CCR7 and CD45RA, indicative of T effector cells, as shown here in Figure 4 and in references3,7. This quality is a central paradox to the system; the immunosuppression allows the growth of allogeneic human tumors in the presence of an allogeneic HIS, at rates similar to or even faster than non-humanized BRGS or nude (Nu/J) mice3,7,23. However, immune stimuli have been shown to be able to overcome this immunosuppression and reject a tumor7,8. Although significant changes in tumor growth between vehicle and treated mice are not always observed, significant differences in immune phenotypes have been found more often, so analyzing the tumor growth rates correlated to immune phenotypes is suggested. In this analysis, immune parameters were found that significantly correlate with reduced tumor growth3. These data suggest that particular &#34;immunocorrelates&#34; are treatment-related and participate in tumor control. Thus, tumor growth measurements combined with immune phenotypes in the HIS mouse model provide important preclinical data with regard to tumor immune responses to unique human tumors. The challenge for any preclinical model is correlation to clinical results. The HIS mouse oncology field has several examples of clinical correlation: 1) we and others have shown that the human immune infiltration correlates with the tumor type3,22,23,40; 2) we have shown successful treatment with anti-PD-1 in an ACC Lynch syndrome PDX taken from a patient who subsequently responded to this therapy31; and 3) we have shown superior responses to a CRC MSS hi PDX, a cancer with high response rates in the clinic over the notoriously poor-responding CRC MSS PDX7. Together with in vitro and other in vivo models, these data provide a nexus for translation into the clinic.

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/t/64606">Testing Cancer Immunotherapeutics in a Humanized Mouse Model Bearing Human Tumors</a>. The Authors section was updated from:
Jordi M. Lanis1 
Matthew S. Lewis1 
Hannah Strassburger1 
Stacey M. Bagby2 
Adrian T. A. Dominguez2 
Juan A. Mar&#237;n-Jim&#233;nez3 
Roberta Pelanda1 
Todd M. Pitts2 
Julie Lang1 
1Department of Immunology and Microbiology, School of Medicine, University of Colorado Denver Anschutz Medical Campus 
2Division of Oncology, School of Medicine, University of Colorado Denver Anschutz Medical Campus 
3Department of Medical Oncology, Catalan Institute of Oncology (ICO-L&#8217;Hospitalet)
to:
Jordi M. Lanis1 
Matthew S. Lewis1 
Hannah Strassburger1 
Kristina Larsen1 
Stacey M. Bagby2 
Adrian T. A. Dominguez2 
Juan A. Mar&#237;n-Jim&#233;nez3 
Roberta Pelanda1 
Todd M. Pitts2 
Julie Lang1 
1Department of Immunology and Microbiology, School of Medicine, University of Colorado Denver Anschutz Medical Campus 
2Division of Oncology, School of Medicine, University of Colorado Denver Anschutz Medical Campus 
3Department of Medical Oncology, Catalan Institute of Oncology (ICO-L&#8217;Hospitalet)

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/t/64606">Testing Cancer Immunotherapeutics in a Humanized Mouse Model Bearing Human Tumors</a>. The Authors section was updated.

Erratum: Testing Cancer Immunotherapeutics in a Humanized Mouse Model Bearing Human Tumors

Mouse cancer models are important for establishing basic mechanisms of tumor growth and immune escape. However, cancer treatment studies in mouse models have yielded finite translation to the clinic due to limited syngeneic models and species-specific differences1,2. The emergence of immune therapies as a dominant approach to control tumors has reiterated the need for an in vivo model with a functional human immune system. Advancements in human immune system mice (HIS mice) over the past decade have made it possible to study immuno-oncology in vivo in a wide variety of cancer types and immunotherapeutic agents3,4,5,6. Human tumor models, including cell-line derived and patient-derived xenografts (CDX and PDX, respectively), grow well in HIS mice and in most cases are nearly identical to their growth in the immunodeficient host lacking human hematopoietic engraftment7,8. Based on this key finding, researchers have been using the HIS mouse model to study human immunotherapies, including combination therapies designed to alter the tumor microenvironment (TME) to decrease immunosuppression and thus enhance immune-directed tumor killing. These preclinical models help address the issues of heterogeneity of human cancers, and can also predict treatment success as well as monitor immune related drug toxicities9,10.
The production of a mouse model with a human immune system through the introduction of human hematopoietic stem cells requires a recipient immunodeficient mouse that will not reject the xenograft. Current HIS mouse models are derived from immunodeficient mouse strains that were reported over 30 years ago. The first immunodeficient mouse strain described was SCID mice that lacked T and B cells11, followed by a hybrid NOD-SCID with an SIRP&#945;&#160;polymorphism responsible for mouse macrophage tolerance to human cells, due to increased binding for the NOD SIRP&#945; allele to the human CD47 molecule12,13. In the early 2000s, the deletion of the common gamma chain of the IL-2 receptor (IL-2R&#947;c) on both BALB/c and NOD immunodeficient strains was a game changer for enhanced human engraftment, due to genetic deletions forbidding host NK cell development14,15,16,17. Alternative models, such as BRG and NRG mice, achieve T and B cell deficiency through deletion of the Rag1 or Rag2 gene, required for T and B cell receptor gene rearrangements and thus the maturation and survival of lymphocytes18,19. The BRGS (BALB/c -Rag2nullIl2R&#947;CnullSirp&#945;NOD) mouse used herein combines the IL-2R&#947; chain deficiency and the NOD SIRP&#945; allele on the Rag2-/- background, resulting in a highly immunodeficient mouse without T, B, or NK cells, yet with sufficient vigor and health to allow for long term engraftment of more than 30 weeks13.
HIS mice can be generated in multiple ways, with human PBMC injection being the most direct method15,18,20. However, these mice have a pronounced expansion of activated human T cells that results in graft versus host disease (GVHD) by 12 weeks of age, preventing long-term studies. Alternatively, human hematopoietic stem cells from umbilical cord blood (CB), bone marrow, and fetal liver can also be used for engraftment and production of the human immune system de novo. In this system, the hematopoietic stem cells produce a multi-lineage human immune system with the generation of T, B, and innate immune cells that are importantly tolerant of the mouse host, compared to the PBMC mice that develop mostly T cells. Therefore, GVHD is absent or greatly delayed, and studies can be extended to mice up to 10 months of age. CB provides an easy, accessible, and noninvasive source of CD34+ human hematopoietic stem cells that facilitates the engraftment of multiple HIS mice with genetically identical immune systems17,18,20,21. Over the past few years, HIS mouse models have been used extensively to study immunotherapy and the TME3,4,5,6. Despite the development of human derived immune systems in these mice, human xenograft tumors grow at similar rates compared to the control immunodeficient mice and allow for the complex interplay between the cancer cells and immune cells, which is important for maintaining the microenvironment of the engrafted PDX3,7,8. This protocol has been used to perform over 50 studies testing treatments in HIS-BRGS mice with PDXs and CDXs. An important conclusion is that human tumors in the HIS mice maintain their unique TME as defined by molecular evaluation of the tumor relative to the initial patient sample and immune infiltrate characteristics3,22,23. Our group focuses on in-depth evaluation of the HIS in both immune organs and the tumor using multi-parameter flow cytometry. Herein, we describe a protocol for the humanization of BRGS mice, evaluation of chimerism, implantation of human tumors, tumor growth measurements, cancer treatment administration, and analysis of the HIS cells by flow cytometry.

<table><tbody><tr><td>1 mL syringe w/needles</td><td>McKesson</td><td>1031815</td><td></td></tr><tr><td>15 mL tubes</td><td>Grenier Bio-One</td><td>188271</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Sigma</td><td>M6250</td><td></td></tr><tr><td>50 mL tubes</td><td>Grenier Bio-One</td><td>227261</td><td></td></tr><tr><td>AutoMACS Pro Separator</td><td>Miltenyi</td><td>130-092-545</td><td></td></tr><tr><td>BD Golgi Stop Protein Transport Inhibitor with monensin</td><td>BD Bioscience</td><td>BDB563792</td><td></td></tr><tr><td>BSA</td><td>Fisher Scientific</td><td>BP1600100</td><td></td></tr><tr><td>Cell Stim Cocktail</td><td>Life Technologies</td><td>509305</td><td></td></tr><tr><td>Chill 15 Rack</td><td>Miltenyi</td><td>130-092-952</td><td></td></tr><tr><td>Cotton-plugged glass pipettes</td><td>Fisher Scientific</td><td>13-678-8B</td><td></td></tr><tr><td>Cultrex Basement membrane extract</td><td>R&amp;amp;D Systems</td><td>363200502</td><td></td></tr><tr><td>Cytek Aurora</td><td>Cytek</td><td></td><td></td></tr><tr><td>DNase</td><td>Sigma</td><td>9003-98-9</td><td></td></tr><tr><td>eBioscience FoxP3/Transcription Factor Staining Buffer Set</td><td>Invitrogen</td><td>00-5523-00</td><td></td></tr><tr><td>Embryonic Stemcell FCS</td><td>Gibco</td><td>10439001</td><td></td></tr><tr><td>Eppendorf Tubes; 1.5 mL volume</td><td>Grenier Bio-One</td><td>616201</td><td></td></tr><tr><td>Excel</td><td>Microsoft</td><td></td><td></td></tr><tr><td>FBS</td><td>Benchmark</td><td>100-106 500mL</td><td></td></tr><tr><td>Ficoll Hypaque</td><td>GE Healthcare</td><td>45001752</td><td></td></tr><tr><td>FlowJo Software</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>Forceps - fine</td><td>Roboz Surgical&amp;nbsp;</td><td>RS5045</td><td></td></tr><tr><td>Forceps normal</td><td>Dumont</td><td>RS4919</td><td></td></tr><tr><td>Formaldehyde</td><td>Fisher</td><td>F75P1GAL</td><td></td></tr><tr><td>Frosted Glass Slides</td><td>Corning</td><td>1255310</td><td></td></tr><tr><td>Gentlemacs C-Tubes</td><td>Miltenyi&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>130-096-334</td><td></td></tr><tr><td>GentleMACS Dissociator</td><td>Miltenyi</td><td>130-093-235</td><td></td></tr><tr><td>glass pipettes</td><td>DWK Life Sciences</td><td>63A53</td><td></td></tr><tr><td>Glutamax</td><td>Gibco</td><td>11140050</td><td></td></tr><tr><td>HBSS w/ Ca &amp;amp; Mg</td><td>Sigma</td><td>55037C</td><td></td></tr><tr><td>HEPES</td><td>Corning</td><td>MT25060CI</td><td></td></tr><tr><td>IgG standard</td><td>Sigma</td><td>I2511</td><td></td></tr><tr><td>IgM standard</td><td>Sigma</td><td>401108</td><td></td></tr><tr><td>IMDM</td><td>Gibco</td><td>12440053</td><td></td></tr><tr><td>Liberase DL</td><td>Roche</td><td>5466202001</td><td></td></tr><tr><td>LIVE/DEAD Fixable Blue</td><td>Thermo</td><td>L23105</td><td></td></tr><tr><td>MDA-MB-231</td><td>ATCC</td><td>HTB-26</td><td></td></tr><tr><td>MEM</td><td>Gibco</td><td>1140050</td><td></td></tr><tr><td>mouse anti-human IgG-AP</td><td>Southern Biotech</td><td>JDC-10</td><td></td></tr><tr><td>mouse anti-human IgG-unabeled</td><td>Southern Biotech</td><td>H2</td><td></td></tr><tr><td>mouse anti-human IgM-AP</td><td>Southern Biotech</td><td>UHB</td><td></td></tr><tr><td>mouse anti-human IgM-unlabeled</td><td>Southern Biotech</td><td>SA-DA4</td><td></td></tr><tr><td>MultiRad 350</td><td>Precision X-Ray</td><td></td><td></td></tr><tr><td>PBS</td><td>Corning</td><td>45000-446</td><td></td></tr><tr><td>Pen Strep</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Petri Dishes</td><td>Fisher Scientific</td><td>FB0875713A</td><td></td></tr><tr><td>p-nitrophenyl substrate</td><td>Thermo</td><td>34045</td><td></td></tr><tr><td>PRISM</td><td>Graphpad</td><td></td><td></td></tr><tr><td>Rec Hu FLT3L</td><td>R&amp;amp;D systems</td><td>308-FK-005/CF</td><td></td></tr><tr><td>Rec Hu IL6</td><td>R&amp;amp;D systems</td><td>206-IL-010/CF</td><td></td></tr><tr><td>Rec Hu SCF</td><td>R&amp;amp;D systems</td><td>255SC010</td><td></td></tr><tr><td>RPMI 1640</td><td>Corning</td><td>45000-39</td><td></td></tr><tr><td>Saponin</td><td>Sigma</td><td>8047-15-2</td><td></td></tr><tr><td>Scissors</td><td>McKesson</td><td>862945</td><td></td></tr><tr><td>Serological pipettes 25 mL</td><td>Fisher Scientific</td><td>1367811</td><td></td></tr><tr><td>Sterile filter</td><td>Nalgene</td><td>567-0020</td><td></td></tr><tr><td>Sterile molecular water</td><td>Sigma</td><td>7732-18-5</td><td></td></tr><tr><td>Yeti Cell Analyzer</td><td>Bio-Rad</td><td>12004279</td><td></td></tr><tr><td>Zombie Green</td><td>biolegend</td><td>423112</td><td></td></tr></tbody></table>

testing cancer immunotherapeutics in a humanized mouse model bearing human tumors

Reversing the immunosuppressive nature of the tumor microenvironment is critical for the successful treatment of cancers with immunotherapy drugs. Murine cancer models are extremely limited in their diversity and suffer from poor translation to the clinic. To serve as a more physiological preclinical model for immunotherapy studies, this protocol has been developed to evaluate the treatment of human tumors in a mouse reconstituted with a human immune system. This unique protocol demonstrates the development of human immune system (HIS, &#34;humanized&#34;) mice, followed by implantation of a human tumor, either a cell-line derived xenograft (CDX) or a patient derived xenograft (PDX). HIS mice are generated by injecting CD34+ human hematopoietic stem cells isolated from umbilical cord blood into neonatal BRGS (BALB/c Rag2-/- IL2R&#947;C-/- NODSIRP&#945;) highly immunodeficient mice that are also capable of accepting a xenogeneic tumor. The importance of the kinetics and characteristics of the human immune system development and tumor implantation is emphasized. Finally, an in-depth evaluation of the tumor microenvironment using flow cytometry is described. In numerous studies using this protocol, it was found that the tumor microenvironment of individual tumors is recapitulated in HIS-PDX mice; &#34;hot&#34; tumors exhibit large immune infiltration while &#34;cold&#34; tumors do not. This model serves as a testing ground for combination immunotherapies for a wide range of human tumors and represents an important tool in the quest for personalized medicine.

Following the flank tumor protocol and experimental timeline (Figure 1), the tumor growth and immune response to a targeted tyrosine kinase inhibitor (TKI) therapy and nivolumab combination treatment was studied in two distinct human colorectal cancer (CRC) PDXs. The TKI drugs have been studied in immunodeficient hosts to evaluate tumor growth only29. This model enabled the study of changes in the immune response of the TKI alone, and more importantly, in combination with anti-PD-1. This study was focused on the combination treated cohorts in two distinct experiments that represent a successful evaluation using HIS-BRGS mice and a technically flawed experiment. For PDX CRC307P, the combination treatment slowed the growth of the tumor, as determined by tumor growth volumes over time, tumor weights at harvest, and the SGR (Figure 2A-C). On the other hand, the growth of a PDX developed from a metastatic tumor from the same patient, PDX CRC307M, tested in a different cohort of HIS-BRGS mice, was less affected by the same combination treatment in HIS-BRGS mice (Figure 2D).
Despite the allocation of HIS-BRGS mice into equivalent experimental groups based on overall human (hCD45+) and human T cell (hCD3+) chimerism in the blood prior to tumor implantation (Figure 3A-C), both parameters increased in the peripheral immune system (spleens) and the tumor-infiltrating leukocytes (TIL) in combination treated CRC307P-bearing mice, but not the CRC307M model (Figure 3D-F). Notably, although both HIS-BRGS cohorts had comparable human and T cell chimerism in the blood prior to tumor implantation, the CRC307M cohort had very little lymph node chimerism and failed to develop appreciable levels of T cells in the spleen in the treated cohort (Figure 3D-F). The CRC307M experiment represents a technically flawed example due to overall insufficient splenic T cell chimerism and lymph node chimerism. We suggest the exclusion of HIS mice that have &#60;20% hCD3+ chimerism in the spleens and/or &#60;1.5 x 106 hCD45+ cells in the lymph nodes at the end of the study. For the CRC307M model, the majority of mice were excluded, mostly due to very small lymph nodes, leaving only two mice per cohort.
Further investigation of the human T cells (Figure 4A) revealed more activated (HLA-DR+) T cells in the CR307P tumors, but not the lymph, from combination-treated mice (Figure 4B). In the CRC307M &#34;negative&#34; experiment, there was no significant increase of HLA-DR+ T cells in the TILs of treated HIS-BRGS mice when analyzing all the mice, suggesting this non-optimal HIS-BRGS cohort influenced the data significance due to HIS mice with very few T cells and no activation (Figure 4B). Indeed, the increase in HLA-DR+ T cells in the TILs in the CRC307M model did reach significance when excluding the low T cell chimerism HIS-BRGS mice (Figure 4B). In addition, there were more effector memory CD8+ T cells and fewer TIM-3+ (terminally exhausted) T cells in the combination-treated CRC307P tumors, whereas this difference was not noted in the CRC307M model (Figure 4C,D). In this experiment, no changes in the frequencies of cytotoxic T cells (Granzyme B+ or IFN&#947;+TNF&#945;+) populations were observed among the combination-treated mice, although higher cytotoxic T cells were observed in untreated (Figure 4E) or treated (data not shown) tumors relative to lymph nodes. Importantly, although there were no differences in frequencies among the T cell populations, higher numbers of cytotoxic T cells were observed in the tumors of treated HIS-CRC307P-BRGS mice, with higher frequencies (Figure 3B) and numbers of human T cells in the tumors. On the other hand, this combination treatment showed no effect on the frequencies of Tregs in either CRC307P lymph organs or tumors, although the CRC307M data showed a trend of reduced Tregs that would need to be validated in another experiment (Figure 4F).
In addition to interrogating the human immune system, immune-related changes on tumor cells were also evaluated using flow cytometry (Figure 5A). By gating on EpCAM+ cells, as described in the protocol, increased expression of both MHC Class I (HLA-ABC) and Class II (HLA-DR) was found on the CRC307P tumor cells excised from combination-treated HIS-BRGS mice (Figure 5B). In the CRC307M model, this same drug treatment induced HLA Class II expression on the tumor cells, although to a lesser degree than in the CRC307P model (Figure 5C). Thus, the combination treatment appears to induce upregulation of MHC class II, independent of T cell infiltration (Figure 5B,C). Notably, MHC expression on the tumor cells was lower than that on human immune cells, a finding consistent with human tumor reports (Figure 5B). Similarly, the combination-treatment resulted in increased PD-L1 expression on the EpCAM+ CRC307P tumor cells (Figure 5D).
Finally, correlations of immune responses with tumor growth were investigated by plotting the SGR of the tumor versus immune parameters. Although the frequency of CD4+ T cells in the tumors of untreated mice showed no correlation with tumor growth, increased CD4+ T cells showed a significant (Figure 6A) correlation with smaller tumor growth, and more specifically the HLA-DR+ activated T cells (Figure 6B), in the combination treated HIS mice.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64606/64606fig01.jpg" /> 
Figure 1: Schematic illustrating the generation of HIS-BRGS mice and implantation of human CDX or PDX tumors for cancer immunotherapy studies. The timeline is important to ensure the presence of T cells that take months to engraft in the CB-derived HIS mice. Created with Biorender.com. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64606/64606fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64606/64606fig02.jpg" /> 
Figure 2: Analysis of tumor growth. Tumor growth measurements for CRC307P PDX in control (black) or combination treated (red) HIS-BRGS mice quantified by (A) volume over time, (B) weights at the end of the study, or (C) specific growth rate (SGR). (D) SGRs for CRC307M PDX. Data include tumors implanted into both flanks of six vehicle HIS-BRGS mice, seven combination-treated BRGS mice for CRC307P, and only two vehicle and combination-treated mice for CRC307M due to high exclusion rates. Statistical analyses between two independent groups were performed using unpaired, parametric two-group Welch&#39;s t-test. **p &#60; 0.01, ****p &#60; 0.0001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64606/64606fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64606/64606fig03.jpg" /> 
Figure 3: Human immune and T cell chimerism in lymph organs and tumors of CRC PDX tumor-bearing HIS-BRGS mice. (A) Representative flow cytometry analysis of human (hCD45) and mouse (mCD45) hematopoietic and human T (CD3) cells in peripheral blood (PBMC) prior to tumor implantation, and in LNs and tumors (TIL) at the end of the study. (B,C) Equivalent human and T cell chimerism in blood at 14 weeks in HIS-BRGS mice subsequently injected with (B) CRC307P or (C) CRC307M PDXs and untreated or treated with combination immunotherapy (Tx). (D-F) Increased human T cells in lymph organs and tumors (TIL) of combination-treated HIS-BRGS-CRC307P mice but not HIS-BRGS-CRC307M mice. Data show human and T cell frequencies on the Y-axis as a percentage of the parent population in (D) lymph nodes (LNs), (E) spleens (SP), and (F) tumors of individual mice at the end of the study. Statistical analyses between two independent groups were performed using unpaired, parametric two-group Welch&#39;s t-test. *p &#60; 0.05, **p &#60; 0.01, ***p &#60; 0.001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64606/64606fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64606/64606fig04.jpg" /> 
Figure 4: Analysis of T cell activation by flow cytometry in peripheral lymph organs and tumors of CRC PDX bearing HIS-BRGS mice. (A) Representative flow cytometry analysis of T cells in LNs, spleens (SPs), and tumors (TILs) excised from HIS-BRGS mice measuring the following populations: activated T cells (HLA-DR+), na&#239;ve T cells (CD45RA+ CCR7+), Tem (CD45RA-, CCR7-), Tregs (CD25+, FoxP3+), and cytotoxic T cells (Granzyme B+, TNF&#945;+, and/or IFN&#947;+). (B-D) Frequencies of indicated T cell populations in lymph organs or TILs of HIS-CRC307P-BRGS and HIS-CRC307M-BRGS mice: (B) HLA-DR+ activated T-cells, (C) CCR7-CD45RA-Tem CD8+ T cells, (D) inhibitory receptor TIM-3 CD8+ T cells, (E) Granzyme B CD8+ T cells, and (F) CD25+FoxP3+ CD4+ Tregs. For the HIS-CRC307M-BRGS mice in (B), the first data set shows all mice analyzed at harvest, whereas the second data set includes only those with sufficient LN and splenic T cell chimerism. In all graphs, the filled symbols for 307M represent mice with sufficient chimerism, whereas open symbols are excluded HIS mice. Statistical analyses between two independent groups were performed using unpaired, parametric two-group Welch&#39;s t-test. *p &#60; 0.05, **p &#60; 0.01, ***p &#60; 0.001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64606/64606fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64606/64606fig05.jpg" /> 
Figure 5: Flow cytometry analysis of MHC class I, II, and PD-L1 on human tumors in HIS-BRGS mice. (A) Representative flow cytometry dot plots, illustrating gating strategy for measuring expression levels of MHC Class I (HLA-ABC), Class II (HLA-DR), and PD-L1 on epithelial (EpCAM+) human tumors in HIS-BRGS mice. (B-D) Mean fluorescence intensity (MFI) of HLA-ABC (B), HLA-DR (B,C), and PD-L1 (D) on human (hCD45+) and tumor (EpCAM+) cells from dissociated CRC307P (B,D) or CRC307M (C) PDXs excised from HIS-BRGS mice. Statistical analyses between two independent groups were performed using unpaired, parametric two-group Welch&#39;s t-test. *p &#60;0.05, **p&#60;0.01, ****p&#60;0.0001. Abbreviation: Tx = Treatment <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64606/64606fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64606/64606fig06.jpg" /> 
Figure 6: Analysis of immunocorrelates of response. The SGRs of CRC307P tumor growth in the flanks of untreated (Veh) or combination immunotherapy-treated (Tx) HIS-BRGS mice were plotted versus the frequency of (A) CD4+ or (B) HLA-DR+ T cells as an indicator of immune parameters that correlate with reduced tumor growth (i.e., smaller SGR). A simple linear regression analysis was performed to indicate significant correlations. R2 values indicate degree of correlation. *p &#60; 0.05. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64606/64606fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Surface staining panel worksheet. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64606/Table 1.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Cell staining panels and flow cytometry gating worksheet. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64606/Table 2.xlsx" target="_blank">Please click here to download this Table.</a>
Supplemental File 1: Recipes for media and solutions used in the study. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64606/Supplemental File 1.xlsx" target="_blank">Please click here to download this File.</a>
Supplemental File 2: Representative flow cytometry gating for each stain. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64606/Supplemental File 2.pdf" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Testing Cancer Immunotherapeutics in a Humanized Mouse Model Bearing Human Tumors at JoVE.com

Testing Cancer Immunotherapeutics in a Humanized Mouse Model Bearing Human Tumors

This protocol outlines the generation of human immune system (HIS) mice for immuno-oncology studies. Instructions and considerations in the use of this model for testing human immunotherapeutics on human tumors implanted in this model are presented with an emphasis on characterizing the response of the human immune system to the tumor.

Reversing the immunosuppressive nature of the tumor microenvironment is critical for the successful treatment of cancers with immunotherapy drugs. Murine ...

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Nanyang Technological University

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JoVE Journal

Cancer Research

3.0K Views.  University of Colorado Denver Anschutz Medical Campus. Dit protocol schetst de generatie van het menselijk immuunsysteem, of HIS-muizen voor preklinische immuno-oncologische studies. Muizenkankermodellen zijn beperkt in hun diversiteit en vertalen zich slecht naar de kliniek. Deze techniek dient als een preklinisch model voor immunotherapiestudies door de behandeling van menselijke tumoren in een muis met een menselijk immuunsysteem te evalueren.Het gehumaniseerde muismodel kan worden gebruikt om meerdere aspecten van de menselijke immunol...

Video: Testing Cancer Immunotherapeutics in a Humanized Mouse Model Bearing Human Tumors

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified to secrete human Prostate Specific Antigen (PSA), and the procedure for the confirmation of tumor implantation. In brief, mice are anesthetized using injectable drugs and are made to lay in the dorsal position. Urine is vacated from the bladder and 50 &#181;L of poly-L-lysine (PLL) is slowly instilled at a rate of 10 &#181;L/20 s using a 24 G IV catheter. It is left in the bladder for 20 min by stoppering the catheter. The catheter is removed and PLL is vacated by gentle pressure on the bladder. This is followed by instillation of the murine bladder cancer cell line (1 x 105 cells/50 &#181;L) at a rate of 10 &#181;L/20 s. The catheter is stoppered to prevent premature evacuation. After 1 h, the mice are revived with a reversal drug, and the bladder is vacated. The slow instillation rate is important, as it reduces vesico-ureteral reflux, which can cause tumors to occur in the upper urinary tract and in the kidneys. The cell line should be well re-suspended to reduce clumping of cells, as this can lead to uneven tumor sizes after implantation.
This technique induces tumors with high efficiency. Tumor growth is monitored by urinary PSA secretion. PSA marker monitoring is more reliable than ultrasound or fluorescence imaging for the detection of the presence of tumors in the bladder. Tumors in mice generally reach a maximum size that negatively impacts health by about 3 - 4 weeks if left untreated. By monitoring tumor growth, it is possible to differentiate mice that were cured from those that were not successfully implanted with tumors. With only end-point analysis, the latter may be mistakenly assumed to have been cured by therapy.

This protocol describes the generation of murine orthotopic bladder tumors in female C57BL/6J mice and the monitoring of tumor growth.

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified ...

A Melanoma Patient-Derived Xenograft Model

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture vessels versus in vivo in human patients. This is due to the bottleneck selection of clonal populations of melanoma cells that can robustly grow in vitro in the absence of physiological conditions. Further, responses to therapy in two-dimensional tissue cultures overall do not faithfully reflect responses to therapy in melanoma patients, with the majority of clinical trials failing to show the efficacy of therapeutic combinations shown to be effective in vitro. Although xenografting of melanoma cells into mice provides the physiological in vivo context absent from two-dimensional tissue culture assays, the melanoma cells used for engraftment have already undergone bottleneck selection for cells that could grow under two-dimensional conditions when the cell line was established. The irreversible alterations that occur as a consequence of the bottleneck include changes in growth and invasion properties, as well as the loss of specific subpopulations. Therefore, models that better recapitulate the human condition in vivo may better predict therapeutic strategies that effectively increase the overall survival of patients with metastatic melanoma. The patient-derived xenograft (PDX) technique involves the direct implantation of tumor cells from the human patient to a mouse recipient. In this manner, tumor cells are consistently grown under physiological stresses in vivo and never undergo the two-dimensional bottleneck, which preserves the molecular and biological properties present when the tumor was in the human patient. Notable, PDX models derived from organ sites of metastases (i.e., brain) display similar metastatic capacity, while PDX models derived from therapy naive patients and patients with acquired resistance to therapy (i.e., BRAF/MEK inhibitor therapy) display similar sensitivity to therapy.

Patient-derived xenograft (PDX) models more robustly recapitulate melanoma molecular and biological features and are more predictive of therapy response compared to traditional plastic tissue culture-based assays. Here we describe our standard operating protocol for the establishment of new PDX models and the characterization/experimentation of existing PDX models.

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture ...

A Human Peripheral Blood Mononuclear Cell (PBMC) Engrafted Humanized Xenograft Model for Translational Immuno-oncology (I-O) Research

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment challenges persist. Robust and disease-relevant animal models are vital resources for continued preclinical research and development in order to address a range of additional immune checkpoints. Here, we describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft model. BGB-A317 (Tislelizumab), an investigational humanized anti-PD-1 antibody in late-stage clinical development, is used as an example to discuss platform set-up, model characterization and drug efficacy evaluations. These humanized mice support the growth of most human tumors tested, thus allowing the assessment of I-O therapies in the context of both human immunity and human cancers. Once established, our model is comparatively time- and cost-effective, and usually yield highly reproducible results. We suggest that the protocol outlined in this article could serve as a general guideline for establishing mouse models reconstituted with human PBMC and tumors for I-O research.

A Human Peripheral Blood Mononuclear Cell PBMC Engrafted Humanized Xenograft Model for Translational Immuno-oncology I-O Research

We describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft mouse model for translational immuno-oncology research. This protocol could serve as a general guideline for establishing and characterizing similar models for I-O therapy assessment.

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment ...

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

Small animal models such as mice have been extensively used to study human disease and to develop new therapeutic interventions. Despite the wealth of information gained from these studies, the unique characteristics of mouse immunity as well as the species specificity of viral diseases such as human immunodeficiency virus (HIV) infection led to the development of humanized mouse models. The earlier models involved the use of C. B 17 scid/scid mice and the transplantation of human fetal thymus and fetal liver termed thy/liv (SCID-hu) 1, 2 or the adoptive transfer of human peripheral blood leukocytes (SCID-huPBL) 3. Both models were mainly utilized for the study of HIV infection.
One of the main limitations of both of these models was the lack of stable reconstitution of human immune cells in the periphery to make them a more physiologically relevant model to study HIV disease. To this end, the BLT humanized mouse model was developed. BLT stands for bone marrow/liver/thymus. In this model, 6 to 8 week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immunocompromised mice receive the thy/liv implant as in the SCID-hu mouse model only to be followed by a second human hematopoietic stem cell transplant 4. The advantage of this system is the full reconstitution of the human immune system in the periphery. This model has been used to study HIV infection and latency 5-8.
We have generated a modified version of this model in which we use genetically modified human hematopoietic stem cells (hHSC) to construct the thy/liv implant followed by injection of transduced autologous hHSC 7, 9. This approach results in the generation of genetically modified lineages. More importantly, we adapted this system to examine the potential of generating functional cytotoxic T cells (CTL) expressing a melanoma specific T cell receptor. Using this model we were able to assess the functionality of our transgenic CTL utilizing live positron emission tomography (PET) imaging to determine tumor regression (9).
The goal of this protocol is to describe the process of generating these transgenic mice and assessing in vivo efficacy using live PET imaging. As a note, since we use human tissues and lentiviral vectors, our facilities conform to CDC NIH guidelines for Biosafety Level 2 (BSL2) with special precautions (BSL2+). In addition, the NSG mice are severely immunocompromised thus, their housing and maintenance must conform to the highest health standards (<a href="http://jaxmice.jax.org/research/immunology/005557-housing.html" target="_blank">http://jaxmice.jax.org/research/immunology/005557-housing.html</a>).

The generation and characterization of tumor specific T cells using humanized mice is described here. Human thymic tissue and genetically modified human hematopoietic stem cells are transplanted into immunocompromised mice. This results in the reconstitution of an engineered human immune system allowing for in vivo examination of anti-tumor immune responses.

Small animal models such as mice have been extensively used to study human  disease and to develop new therapeutic interventions. Despite the wealth of  ...

Immunology and Infection

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in eradicating cancerous cells. However, the origins and replenishment of tumor antigen-specific CD8+ T cells within the tumor microenvironment (TME) remain obscure. This protocol employs the B16F10-OVA melanoma cell line, which stably expresses the surrogate neoantigen, ovalbumin (OVA), and TCR transgenic OT-I mice, in which over 90% of CD8+ T cells specifically recognize the OVA-derived peptide OVA257-264 (SIINFEKL) bound to the class I major histocompatibility complex (MHC) molecule H2-Kb. These features enable the study of antigen-specific T cell responses during tumorigenesis.
Combining this model with tumor transplantation surgery, tumor tissues from donors were transplanted into tumor-matched syngeneic recipient mice to precisely trace the influx of recipient-derived immune cells into transplanted donor tissues, allowing the analysis of the immune responses of tumor-inherent and periphery-originated antigen-specific CD8+ T cells. A dynamic transition was found to occur between these two populations. Collectively, this experimental design has provided another approach to precisely investigate the immune responses of CD8+ T cells in TME, which will shed new light on tumor immunology.

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

Here, we present a tumor transplantation protocol for the characterization of tumor-inherent and periphery-derived tumor-infiltrated lymphocytes in a mouse tumor model. Specific tracing of the influx of recipient-derived immune cells with flow cytometry reveals the dynamics of the phenotypic and functional changes of these cells during antitumor immune responses.

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in ...

Assessment of Chimeric Antigen Receptor T Cell-Associated Toxicities Using an Acute Lymphoblastic Leukemia Patient-Derived Xenograft Mouse Model

Chimeric antigen receptor T (CART) cell therapy has emerged as a powerful tool for the treatment of multiple types of CD19+ malignancies, which has led to the recent FDA approval of several CD19-targeted CART (CART19) cell therapies. However, CART cell therapy is associated with a unique set of toxicities that carry their own morbidity and mortality. This includes cytokine release syndrome (CRS) and neuroinflammation (NI). The use of preclinical mouse models has been crucial in the research and development of CART technology for assessing both CART efficacy and CART toxicity. The available preclinical models to test this adoptive cellular immunotherapy include syngeneic, xenograft, transgenic, and humanized mouse models. There is no single model that seamlessly mirrors the human immune system, and each model has strengths and weaknesses. This methods paper aims to describe a patient-derived xenograft model using leukemic blasts from patients with acute lymphoblastic leukemia as a strategy to assess CART19-associated toxicities, CRS, and NI. This model has been shown to recapitulate CART19-associated toxicities as well as therapeutic efficacy as seen in the clinic.

Here, we describe a protocol in which an acute lymphoblastic leukemia patient-derived xenograft model is used as a strategy to assess and monitor CD19-targeted chimeric antigen receptor T cell-associated toxicities.

Chimeric antigen receptor T (CART) cell therapy has emerged as a powerful tool for the treatment of multiple types of CD19+ malignancies, which has led to ...

Chronic, Acute, and Reactivated HIV Infection in Humanized Immunodeficient Mouse Models

Humanized NOD/SCID/IL-2 receptor &#947;-chainnull mice recapitulate some features of human immunity, which can be exploited in basic and pre-clinical research on infectious diseases. Described here are three models of humanized immunodeficient mice for studying the dynamics of HIV infection. The first is based on the intrahepatic injection of CD34+ hematopoietic stem cells in newborn mice, which allows for the reconstitution of several blood and lymphoid tissue-confined cells, followed by infection with a reference HIV strain. This model allows monitoring for up to 36 weeks post-infection and is hence called the chronic model. The second and third models are referred to as the acute and reactivation models, in which peripheral blood mononuclear cells are intraperitoneally injected in adult mice. In the acute model, cells from a healthy donor are engrafted through the intraperitoneal route, followed by infection with a reference HIV strain. Finally, in the reactivation model, cells from an HIV-infected donor under antiretroviral therapy are engrafted via the intraperitoneal route. In this case, a drug-free environment in the mouse allows for virus reactivation and an increase in viral load. The protocols provided here describe the conventional experimental approach for humanized, immunodeficient mouse models of HIV infection.

Described here are three experimental approaches for studying the dynamics of HIV infection in humanized mice. The first permits the study of chronic infection events, whereas the two latter allows for the study of acute events after primary infection or viral reactivation.

Humanized NOD/SCID/IL-2 receptor &#947;-chainnull mice recapitulate some features of human immunity, which can be exploited in basic and pre-clinical ...

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34+ Cells Injection, and Cytokine Delivery

Animal models provide a vital translation between in vitro and in vivo biomedical research. Humanized mouse models provide a bridge in the representation of human systems, thereby allowing for a more accurate study of pathogenesis, biomarkers, and many other scientific queries. In this method described, immune-deficient NOD-scid IL2R&#947;null (NSG) mice are implanted with autologous thymus, injected with liver-derived CD34+ cells followed by a series of injected cytokine deliveries. In contrast to other models of a similar nature, the model described here promotes an improved reconstitution of immune cells by delivering cytokines and growth factors via transgenes encoded in AAV8 or pMV101 DNA-based vectors. Moreover, it offers long-term stability with reconstituted mice having an average lifespan of 30 weeks after CD34+ injections. Through this model, we hope to provide a stable and impactful method of studying immunotherapy and human disease in a murine model, thus demonstrating the need for predictive preclinical models.

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34+ Cells Injection, and Cytokine Delivery

Humanized mouse models provide a more accurate representation of the human immune microenvironment. This manuscript describes the process in which these models are created through a renal graft of human thymus, injection of human CD34+ cells, and the targeted delivery of human cytokine transgenes to promote CD34+ cell proliferation and differentiation.

Animal models provide a vital translation between in vitro and in vivo biomedical research. Humanized mouse models provide a bridge in the representation ...

Tumor Implantation and Injection of a Tumor Vaccine in a Mouse Model

Source: Liu, H. Y., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/64082">Experimental Melanoma Immunotherapy Model Using Tumor Vaccination with a Hematopoietic Cytokine.</a> J. Vis. Exp. (2023).
This video demonstrates a cell-based tumor vaccination assay. A mouse, intradermally injected with B16-F10 melanoma cells for tumor formation, is vaccinated with B16-F10 cells secreting Flt3L cytokine near the developed tumor. The secreted Flt3L from the vaccine promotes the maturation and proliferation of dendritic cells that then present tumor antigens to T cells in lymph nodes, triggering a targeted anti-tumor immune response.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
All mice ...

JoVE Encyclopedia of Experiments

Immunology

Immunotherapy

Immunogenics and Vaccine Development

A Technique to Generate a Human PBMC-Engrafted Humanized Xenograft Mouse Model

Source: Li, Z., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/59679">A Human Peripheral Blood Mononuclear Cell (PBMC) Engrafted Humanized Xenograft Model for Translational Immuno-oncology (I-O) Research.</a> J. Vis. Exp. (2019)
This video demonstrates an assay to generate a humanized xenograft mouse model. An immunocompromised mouse is injected with cyclophosphamide to cause immune cell depletion. Patient-derived tumor cells mixed with human peripheral blood mononuclear cells (PBMCs) in a hydrogel are subcutaneously injected into the mouse, forming a tumor with infiltrating T cells.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines. All procedures involving animal ...

Testing Cancer Immunotherapeutics in a Humanized Mouse Model Bearing Human Tumors

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