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ORIGINAL ARTICLE
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Microsatellite instability-high colorectal cancer patient-derived xenograft models for cancer immunity research


1 Department of Medicine, Division of Medical Oncology/Hematology, Kobe University Hospital and Graduate School of Medicine, Kobe, Japan
2 Department of Medicine, Division of Medical Oncology/Hematology, Kobe University Hospital and Graduate School of Medicine; Cancer Center, Kobe University Hospital, Kobe, Japan
3 Department of Clinical Laboratory, Kobe University Hospital, Kobe, Japan
4 Department of Medicine, Division of Gastroenterology, Kobe University Hospital and Graduate School of Medicine, Kobe, Japan
5 Department of Surgery, Division of Gastrointestinal Surgery, Kobe University Hospital and Graduate School of Medicine, Chuo-ku, Kobe, Japan

Date of Submission01-Aug-2020
Date of Decision20-Jan-2021
Date of Web Publication08-Sep-2021

Correspondence Address:
Yohei Funakoshi,
Department of Medicine, Division of Medical Oncology/Hematology, Kobe University Hospital and Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, 650-0017
Japan
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_1092_20

 > Abstract 


Context: There is an increasing demand for appropriate preclinical mice models for evaluating the efficacy of cancer immunotherapies.
Aims: Therefore, we established a humanized patient-derived xenograft (PDX) model using microsatellite instability-high (MSI-H) colorectal cancer (CRC) tissues and patient-derived peripheral blood mononuclear cells (PBMCs).
Subjects and Methods: The CRC tissues of patients scheduled for surgery were tested for MSI status, and CRC tumors were transplanted into NOD/LtSz-scid/IL-2Rg-/-(NSG) mice to establish MSI-H PDX models. PDX tumors were compared to the original patient tumors in terms of histological and genetic characteristics. To humanize the immune system of MSI-H PDX models, patient PBMCs were injected through the tail vein.
Results: PDX models were established from two patients with MSI-H CRC; one patient had a germline mutation in MLH1 (c.1990-2A > G), and the other patient had MLH1 promoter hypermethylation. PDX with the germline mutation was histologically similar to the patient tumor, and retained the genetic characteristics, including MSI-H, deficient mismatch repair (dMMR), and MLH1 mutation. In contrast, the histological features of the other PDX from a tumor with MLH1 promoter hypermethylation were clearly different from those of the original tumor, and MLH1 promoter hypermethylation and MSI-H/dMMR were lost in the PDX. When T cells from the same patient with MLH1 mutation were injected into the PDX through the tail vein, they were detected in the PDX tumor.
Conclusions: The MSI-H tumor with an MMR mutation is suitable for MSI-H PDX model generation. The PBMC humanized MSI-H PDX has the potential to be used as an efficient model for cancer immunotherapy research.

Keywords: Cancer immunity research, germline mutation, microsatellite instability, patient-derived xenograft models, sporadic methylation



How to cite this URL:
Suto H, Funakoshi Y, Nagatani Y, Imamura Y, Toyoda M, Kiyota N, Matsumoto H, Tanaka S, Takai R, Hasegawa H, Yamashita K, Matsuda T, Kakeji Y, Minami H. Microsatellite instability-high colorectal cancer patient-derived xenograft models for cancer immunity research. J Can Res Ther [Epub ahead of print] [cited 2021 Dec 5]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=325758




 > Introduction Top


Immunotherapy using immune checkpoint inhibitors (ICPis) has led to substantial clinical success with durable responses and prolonged survival in cancer patients.[1],[2] Despite these promising results, immunotherapy is not effective for all cancer patients; indeed, the response rate of ICPi monotherapy is only around 20%.[3],[4] Hence, further studies of cancer immunity and immunotherapy are needed. However, because current experimental models, especially animal models, are not fully able to recapitulate human cancer immunity, it is difficult to perform fundamental and preclinical studies, such as biomarker studies and drug efficacy assessments, for immunotherapy.[5],[6]

Patient-derived xenograft (PDX) models have been developed and used as preclinical models for cancer research.[7],[8],[9],[10] PDX models are established following the direct transfer of fresh patients' tumors into severely immunodeficient mice.[7] The ability of PDX models to maintain the original features of patient tumors and to reflect drug sensitivity has improved both basic and clinical study outcomes.[7],[8],[11] However, in terms of cancer immunity research, conventional PDX models are not suitable for use as preclinical models.[12] First, as is clear from the low response rate of ICPi therapy, the majority of tumors are “cold tumors“, defined as those with insufficient immunogenicity and low immune cell infiltrates.[13] Therefore, even if tumors are transplanted at random to establish PDX models, the majority of models are unsuitable for cancer immunity research. Second, because PDX models are established using immunodeficient mice to reduce the rejection of original patient tumors by the mouse immune system, functional immune systems of PDX models are very limiting,[7] and human immune systems do not exist in PDX models.[7]

Recent studies have revealed that cancer patients with microsatellite instability-high (MSI-H) tumors have a significantly higher response rate and tend to achieve a durable response from ICPi, regardless of the cancer type.[14] MSI is a condition of genetic hypermutability (predisposition to mutation) that results from an impaired DNA mismatch repair (MMR) system.[15] MSI-H is characterized by deficient MMR (dMMR) proteins resulting from somatic inactivation, most commonly methylation of the MLH1 (one of the MMR genes) promoter region, or a germline mutation in the MMR gene (MLH1, PMS2, MSH2, or MSH6), known as Lynch syndrome.[15],[16],[17] Because increased MSI creates neoepitopes responsible for the immune response, the number of infiltrating lymphocytes has been reported to be increased in tumors with MSI-H,[14],[15],[18] which has led to MSI-H tumors being termed high immunogenicity tumors called “hot tumors.” Hence, MSI-H tumors are appropriate primary tumor candidates for PDX models for cancer immunity research.[13]

To overcome the lack of human immune system in PDX models, the generation of immune system-humanized PDX models has been attempted. Some research groups have reported that immune-deficient mice engrafted with human CD34+ hematopoietic stem cells (HSCs) demonstrated human multilineage hematopoiesis.[19],[20] Hence, CD34+ humanized mice models appeared to be promising tools to study the crosstalk between human cancer and immune cells. However, because these T cells which are differentiated from CD34+ HSC undergo a murine thymic education, human leukocyte antigen (HLA) restriction between T cells and transplanted tumor cells does not function as expected.[21] In addition, CD34+ HSC and tumor are generally derived from different persons. Therefore, CD34+ HSC humanized PDX models might not be appropriate for use in cancer immunity research.

Our research group suggests that humanized PDX models with human peripheral blood mononuclear cells (PBMCs) collected from the patient who provided the primary tumor are suitable for cancer immunity research. Since both immune cells and tumor cells are from the same patient, HLA restriction is expected to occur normally. We believe that human PBMC-driven humanized models might represent a potential model for cancer immunity research.

In the current study, we first performed MSI tests on colorectal cancer (CRC) patients before surgery and transplanted MSI-H tumors into immunodeficient (NOD/LtSz-scid/IL-2Rg-/-; NSG) mice to establish MSI-H PDX models. We then evaluated whether the PDX tumors maintained the histological and genetic characteristics of the original patient tumors. Finally, we injected patient PBMCs into the MSI-H PDX model to generate a humanized PDX model for cancer immunity research.


 > Subjects and Methods Top


Procurement of patient samples

CRC tissues (biopsy samples) of patients scheduled for surgery were tested for MSI status at Kobe University Hospital between January 2017 and December 2018. Three patients (two MSI-H patients and one microsatellite stable [MSS] patient) underwent surgery, and their tumor tissues were obtained.

Peripheral blood samples were collected from CRC patients who provided primary tumor tissues for PDX models into heparin-containing tubes. PBMCs were separated from peripheral blood samples by Ficoll-Paque Plus using SepMate-50 (STEMCELL Technologies).

DNA extraction from formalin-fixed paraffin-embedded tissue samples

DNA samples were obtained from FFPE CRC tissues using ReliaPrep™ FFPE gDNA Miniprep System (Promega). If viable tumor comprised <30% of the designated tissue, manual microdissection was performed.

MSI status analysis

For MSI analysis, DNA samples obtained from FFPE CRC blocks were analyzed using a panel of mononucleotide repeats (BAT25, BAT26, NR21, NR24, and NR27).[22] The five markers were coamplified in one tube using an AmpliTaq Gold™ 360 Master Mix (Thermo Fisher Scientific). The amplified products were separated on an ABI Prism 3500DX Genetic Analyzer (Thermo Fisher Scientific).[23] MSI-H was defined when 2 or more loci showed instability, MSI-L was defined when 1 locus showed instability, and MSS was defined when no loci showed instability.[23]

Establishment of patient-derived xenograft models

Tissue samples were cut into 1 mm × 1 mm × 1 mm pieces and subcutaneously transplanted into NSG mice. When the tumor size in the implanted area reached an approximate volume of 1500 mm3, the tumors were harvested (P0 generation) for transplantation into the next generation of mice.

Patient PBMCs (1 × 107 cells) were intravenously administered to MSI-H PDX models via the tail vein. For engrafted human immune cell monitoring, peripheral blood samples and PDX tumors were obtained from sacrificed mice 3 days after transplantation. PDX tumors were investigated using immunohistochemistry (IHC) staining.

Histological analysis

FFPE blocks were cut into 4-μm sections. These sections were processed for staining with hematoxylin and eosin (H and E) or IHC analyses. IHC analysis was performed to detect the level of expression of MLH1 (diluted 1:50, Dako), PMS2 (diluted 1:40, Dako), MSH2 (diluted 1:50, Dako), MSH6 (diluted 1:50, Dako), CD45 (diluted 1:300, CELL MARQUE), and CD3 (diluted 1:300, Dako).

Nested polymerase chain reaction and methylation-specific polymerase chain reaction

The methylation status of the promoter regions of MLH1 was determined by methylation-specific (MSP) polymerase chain reaction (PCR), modified as a nested two-step approach for increased sensitivity. DNA extracted from FFPE (1 μg) was subjected to bisulfite conversion and subsequent purification, which were performed according to the EpiTect Fast DNA Bisulfite Kit (Qiagen) protocols.

Approximately 50–100 ng of bisulfite-treated DNA was used for step one of nested MSP. The step one primers, which flanked the CpG-rich regions within the promoters of MLH1, amplified the bisulfite-treated DNA regardless of the methylation status. The PCR products of step one were diluted 1:1000 and subjected to the second step of MSP, which incorporated one set of primers. Step two primers were designed to recognize bisulfite-induced modifications of unmethylated or methylated cytosines. [Additional Table 1] lists the primer sequences and PCR product sizes for this nested MSP approach. PCR was performed using a Thermal Cycler Dice Real Time System (TaKaRa). The PCR conditions for the first step of the nested MSP were as follows: 95°C hot start 10 min, then 40 repetitive cycles of denaturation (95°C x 30 s), annealing (56°C × 30 s), extension (72°C × 30 s), and a final 7 min extension at 72°C. The second step of the nested MSP was performed under the same conditions.



Genomic sequencing

The DNA of the MLH1 promotor region was amplified from the extracted DNA samples by PCR as described above (the first step of the nested MSP). The PCR products were purified and subjected to bidirectional sequencing using an ABI Prism 3130XL (Thermo Fisher Scientific).

To search for germline mutations of MLH1, PMS2, MSH2, and MSH6, DNA sequencing of MLH1, PMS2, MSH2, and MSH6 was performed.

Short tandem repeat analysis

DNA samples from the patient 2 tumor and the PDX 2 tumor were amplified with GenePrint 10 System (Promega), which simultaneously amplifies nine autosomal short tandem repeat (STR) loci (D21S11, TH01, TPOX, vWA, CSF1PO, D16S539, D7S820, D13S317, and D5S818) and an amelogenin marker. STR typing was carried out according to the manufacturer's instructions. The identity of the two tumors (patient 2 tumor and PDX 2 tumor) was expressed as an evaluation value (EV) calculated as EV = (number of coincident peaks of STR profiles between patient 2 tumor and PDX 2 tumor) × 2/(total number of STR profile peaks in patient 2 tumor and PDX 2 tumor).

Ethics statements

All human samples were obtained from, and all genomic analyses were tested on, patients who provided written informed consent for this investigation. The investigation was approved by the Kobe University Hospital Ethics Committee (no. 170144), and the study was conducted in accordance with the Declaration of Helsinki and Japanese Ethical Guidelines for Human Genome/Gene Analysis Research. In terms of animal studies, all animals were cared for and treated according to the Guidelines on Scientific and Ethical Care and Use of Laboratory Animals. All experimental procedures were approved by the Animal Care and Use Committee of Kobe University.


 > Results Top


Characteristics of patients and harvested tumors for PDX models

Two surgically removed MSI-H CRC tissues and one MSS CRC tissue from each of the three patients (MSI-H; patient 1 and patient 2, MSS; patient 3) were transplanted into NSG mice. No patients were treated with chemotherapy or radiation therapy before the surgery. All tumor samples were obtained from primary sites. The clinical characteristics and family trees of the donor patients are shown in [Table 1] and [Additional Figure 1]. The histological and genomic statuses of the harvested tumors are shown in [Table 2].
Table 1: Patient characteristics

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Table 2: Histological and genetic characteristics of the patient tumors and patient-derived xenograft tumors

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Case 1: Patient-derived xenograft model from microsatellite instability-high colorectal cancer caused by a germline mutation in MLH1

The “PDX model from MSI-H CRC of patient 1” (PDX 1) was established and passaged 6 times from one mouse to the next. The H and E staining results indicate that PDX 1 tumor (passage 3 [P3, [Figure 1]a] and passage 6 [P6, [Additional Figure 2]a]) exhibited histological features consistent with those of the original patient 1 tumor, including histological grade (well differentiated) [Table 2], [Figure 1]a, and [Additional Figure 2]a]. Five microsatellite markers (BAT25, BAT26, NR21 NR24, and NR27) were tested to confirm that MSI status was maintained in the PDX model. The PDX 1 tumor (P3) demonstrated instability in all five markers, as demonstrated in the original patient 1 tumor [Table 2]. IHC testing of MMR (MLH1, PMS2, MSH2, and MSH6) proteins showed loss of MLH1 and PMS2 expression in both the original patient 1 and the PDX 1 tumor (P3 and P6) [Table 2] and [Figure 1]b and [Additional Figure 2]b.
Figure 1: H and E and immunohistochemistry staining of patient 1 tumor and third-passage patient-derived xenograft 1 tumor. (a) Representative H and E-stained sections of the patient 1 tumor and the third-passage patient-derived xenograft 1 tumor are shown (×4 and × 20). (b) Expressions of MLH1, PMS2, MSH2, and MSH6 by immunohistochemistry staining are shown in the patient 1 tumor and the third-passage patient-derived xenograft 1 tumor (×4 and × 20)

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According to the Amsterdam criteria II (AMS II) and the revised Bethesda guideline (rBG), Lynch syndrome was suspected as a potential diagnosis of patient 1, and a mutation in MLH1 (c. 1990-2A>G) was detected in the PBMC of patient 1 as well as the PDX 1 tumor (P3) [Figure 2].
Figure 2: Electropherograms of the Sanger sequencing of MLH1 from the peripheral blood of patient 1 and patient-derived xenograft 1 tumor. (a) A heterozygous germline mutation (c.1990-2A > G) in MLH1 was discovered by Sanger sequencing in the peripheral blood of patient 1. (b) The same mutation (c.1990-2A > G) was discovered by Sanger sequencing of the patient-derived xenograft 1 tumor

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These results indicated that PDX 1 tumor maintained the histological and genomic characteristics of the original patient 1 tumor.

Case 2: Patient-derived xenograft model from microsatellite instability-high colorectal cancer caused by a sporadic hypermethylation in a promoter region of MLH1

The “PDX model from MSI-H CRC of patient 2” (PDX 2) was established and passaged 4 times. Unlike case 1, the histological features of PDX 2 tumors (P3 and P4) were clearly different from the original patient 2 tumor [Figure 3]a and [Additional Figure 3]a. Although the patient 2 tumor was diagnosed as well differentiated adenocarcinoma, the PDX 2 tumor was classified as undifferentiated carcinoma with no glandular structure [Table 2] and [Figure 3]a and [Additional Figure 3]a. Next, we evaluated the genetic characteristics of the PDX 2 tumor. Surprisingly, despite the fact that the patient 2 tumor was MSI-H, the PDX 2 tumor (P3) was MSS [Table 2] and [Figure 4]. Consistent with the MSI status, although the patient 2 tumor presented dMMR (IHC loss of MLH1 and PMS2), the PDX 2 tumor (P3 and P4) presented MMR proficient [Figure 3]b and [Additional Figure 3]b.
Figure 3: H and E and IHC staining of the patient 2 tumor and the third-passage patient-derived xenograft 2 tumor. (a) Representative H and E-stained sections of the patient 2 tumor and the third-passage patient-derived xenograft 2 tumor are shown. Magnification ×4 and ×20. (b) Expressions of MLH1, PMS2, MSH2, and MSH6 by IHC staining are shown in the patient 2 tumor and the third-passage patient-derived xenograft 2 tumor. Magnification ×4 and x20

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Figure 4: Testing MSI with five satellite markers using the mononucleotide repeats panel (BAT25, BAT26, NR21, NR24, and NR27). The result of MSI detection in the patient 2 tumor was MSI-H. The mutation loci were BAT25, BAT26, and NR27. The result of MSI detection in the third-passage patient-derived xenograft 2 tumor was MSS

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According to clinical characteristics (unmet AMS II and rBG) and dMMR status, sporadic CRC resulting from MLH1 promoter hypermethylation was suspected as a potential diagnosis of patient 2. Bisulfite-converted DNA sequencing confirmed that the patient 2 tumor had hypermethylation of the MLH1 promoter region [[Figure 5]a, upper]. Next, we analyzed the methylation status of the PDX 2 tumor. Interestingly, no methylation was detected by bisulfite sequencing [[Figure 5]a, bottom]. To support the result of bisulfite sequencing, MSP of the MLH1 promoter region was performed, and the methylation level in the PDX 2 tumor was remarkably decreased compared to that in the patient 2 tumor [Figure 5]b.
Figure 5: Results for bisulfite sequencing and real-time MSP-DNA extracted from the patient 2 tumor and the patient-derived xenograft 2 tumor were bisulfite treated. (a) DNAs were sequenced by Sanger sequencing. Although methylated CpGs were detected in the MLH1 promotor region of the patient 2 tumor, these were not observed in the patient-derived xenograft 2 tumor. (b) The methylation level in the MLH1 promotor region by real-time MSP of the patient-derived xenograft 2 tumor was clearly reduced compared to that of the patient 2 tumor. The MSP primer amplified a band of 115 bp in the DNA from the patient 2 tumor, while no band from the patient-derived xenograft 2 tumor

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Since there were considerable differences in histological and genomic characteristics between the original patient 2 tumor and the PDX 2 tumor, STR analysis was performed to confirm the identification of two tumors. The high EV of STR analysis (83.84%) indicated that the two tumors had the same origin [Additional Figure 4].



Case 3: Patient-derived xenograft model from microsatellite stable colorectal cancer

The “PDX model from MSS CRC of patient 3” (PDX 3) was established as a control model. The PDX 3 tumor was passaged six times, and the tumor maintained the histological and genomic (MSS and MMR status) characteristics of the original patient 3 tumor [Table 2] and [Figure 6] and [Additional Figure 5].
Figure 6: H and E and IHC staining images of the patient 3 tumor and the third-passage patient-derived xenograft 3 tumor. (a) Representative H and E-stained sections of the patient 3 tumor and the third-passage patient-derived xenograft 3 tumor are shown (×4 and × 20). (b) Expressions of MLH1, PMS2, MSH2, and MSH6 by IHC staining are shown in the patient 3 tumor and the third-passage patient-derived xenograft 3 tumor (×4 and × 20)

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Patient 1 T cells infiltrate in the patient-derived xenograft 1 tumor

To confirm whether immune cells from patient 1 are able to infiltrate the PDX 1 tumor, PBMCs from patient 1 were injected into the PDX 1 tumor via the tail vein [Figure 7]a. Some human CD45+ and CD3+ T cells were detected in the PDX 1 tumor 3 days after transplantation [Figure 7]b.
Figure 7: Patient 1 peripheral blood mononuclear cell infiltrate in the MSI-H patient-derived xenograft tumor. (a) Experimental design of peripheral blood mononuclear cell-driven humanized MSI-H patient-derived xenograft models. (b) Patient-derived xenograft 1 was injected with peripheral blood mononuclear cells (1 × 107 cells) from patient 1 via the tail vein. At 3 days after transplantation, patient-derived xenograft 1 tumors were obtained from sacrificed mice, and stained for immunohistochemistry (×20)

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 > Discussion Top


To establish a promising preclinical animal model for cancer immunity research, we first established PDX models using MSI-H tumors. MSI-H/dMMR tumors have been well reported in several types of cancer, most frequently in colorectal, endometrial, and gastric cancer.[24],[25] Although the majority of CRCs develop through chromosomal instability, approximately 10% arise from a MSI pathway that is a consequence of dMMR.[26],[27] Furthermore, given that the engraftment ratio in PDX tumors of CRC are high (70%),[28] we chose CRC as a promising cancer type for MSI-H PDX models. MSI-H CRCs are generally caused by either germline mutations in the MMR gene or sporadic hypermethylation in a promoter region of MLH1.[15],[16],[17] Although there are some phenotypical differences between hereditary and sporadic MSI-H CRC, both MSI-H CRCs are characterized by high immunogenicity resulting in increased T cell infiltration.[29] In addition, recent data have demonstrated that MSI-H CRCs are more likely to respond to ICPi compared to MSS CRC.[14] Therefore, we decided to collect MSI-H CRC regardless of the genetic background, as original primary tumors for MSI-H PDX models.

Although some studies have sought to establish PDX models from MSI-H tumors, research focusing on the change in genetic status resulting in MSI-H is limited.[30],[31],[32] One research group reported that a PDX model from dMMR adrenocortical cancer resulted from a germline mutation in the MSH6 gene.[32] They confirmed that dMMR and MSH6 gene mutation were maintained in PDX model and revealed that the genetic characteristics were maintained.[32] The results of the current study also demonstrate that the MSI-H/dMMR and germline MMR mutation were maintained from the original patient 1 tumor to the PDX 1 tumor.

Whether sporadic hypermethylation is maintained in PDX models is controversial. Another research group reported on three PDX models from MSI-H patient tumors: one with MLH1 promotor hypermethylation and two with MMR mutations (MLH1 and MSH6).[31] Although the detailed characteristics of both patients and PDX tumors were not described, it was demonstrated that the MSI-H status was maintained in the three PDX models, including MLH1 promotor hypermethylation;[31] unlike this previous report, MSI-H/dMMR were not maintained in our study. Importantly, we revealed that hypermethylation of the MLH1 promoter region was not inherited in the NSG mouse. DNA methylation is a biological process through which methyl groups are added to DNA molecules.[33] Promoter DNA methylation-associated CpG islands represents one of the several epigenetic mechanisms that cells use to suppress gene expression,[33] while demethylation activates gene expression.[34] Although it has been reported that demethylation occurs early in embryogenesis in the nuclei of the primordial germ cells,[34] the demethylation mechanism in PDX tumors has not yet been reported. Based on our study, because MSI-H tumors from MLH1 promotor methylation might alter the MSI status after transplantation, we suggest that MSI-H tumors arising from germline mutations are more promising as original primary tumors for MSI-H PDX models. As a study limitation, only 1 model was established in each situation. To clarify our suggestion, further research using multiple models is necessary.

As mice models for cancer immunity research, humanized mice models transplanted with both human PBMC and tumors have been used in some studies.[35],[36] These models are unsuitable for long-term research, because mice harbor human immune cells and develop graft versus host diseases (GVHD). However, since the onset of GVHD is 2–3 weeks post transplantation, the relationship between human immune and cancer cells can be reconstructed in this model and can be observed for short term.[35],[36] Indeed, we were able to detect some human lymphocytes in the PDX 1 tumor without GVHD signs [Figure 7]b.

In summary, we established an MSI-H PDX model (PDX 1) using MSI-H CRC caused by a germline mutation in MLH1. PDX 1 was humanized by engraftment of patient 1 PBMC. Finally, we detected patient 1 CD3+ T cells in the PDX 1 tumor. We suggest that the PBMC humanized MSI-H PDX model is a potential model for use in cancer immunity research.

Financial support and sponsorship

Japanese Society of Medical Oncology (JSMO) under Grant Number 201704 JSPS KAKENHI Grant Number JP18K15319.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
    Tables

  [Table 1], [Table 2]



 

 
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