Abstract
Background
Patient-derived xenografts (PDX) models have been regarded as an important tool for preclinical research. The aim of this study was to establish a Chinese PDX library from gastrointestinal cancers, especially esophageal squamous cell carcinoma (ESCC), esophagogastric junction adenocarcinoma (EGJAC), and gastric adenocarcinoma (GAC).
Methods
1001 surgical tissues or endoscopic biopsy tissues of gastrointestinal cancers were subcutaneously implanted into NOD/SCID mice between January 2013 and August 2015. Engraftment rates, latency period of xenograft formation, patients’ clinicopathological characteristics and survival associated with xenografts for ESCC, EGJAC and GAC were assessed.
Results
208 PDX models were established (20.8%, 208/1001), among which 82 were from ESCC (21.2%, 82/386), 31 from EGJAC (16.9%, 31/183), and 29 from GAC (10.9%, 29/266). The average latency period of xenograft formation of ESCC, EGJAC, and GAC was 76.2, 90.5, and 85.2 days, respectively, for the first passage, and decreased to 52.5, 54.8, and 52.6 days, respectively for the second passage. For ESCC, gender, specimen type and differentiation were associated with engraftment; and for GAC, the factors associated with engraftment were age, specimen type, differentiation, and Lauren classification. The median follow-up of patients with ESCC, EGJAC and GAC was 46, 64 and 64 months, respectively. For GAC, the survival time of patients from whom the tumor tissues achieved successful engraftment was significantly shorter than that without xenograft formation.
Conclusions
We established a Chinese PDX library from gastrointestinal cancers, especially ESCC, a characteristic tumor type in China, providing a platform for drug development and individualized therapy.
Keywords: Patient-derived xenograft model, Esophageal squamous cell carcinoma, Esophagogastric junction adenocarcinoma, Gastric adenocarcinoma
Background
In China, the incidence and mortality of gastrointestinal cancers have been among the top of all cancers [1, 2]. Partially, due to the lack of specific symptoms at early stage, the vast majority of patients with gastrointestinal cancers are diagnozed at advanced stages. For treatment of gastrointestinal cancer, progress has been made from the combination of surgery and chemoradiotherapy, to targeted therapies and immunotherapies in recent years [3–7]. Nonetheless, prognosis of advanced-stage patients is still poor. Therefore, it is urgent to continue advancing anti-cancer drug development and individualized therapy.
In the process of novel anti-cancer therapeutics, preclinical research is the crucial step before clinical trial. Compared with cell lines and cell line-based xenograft models previously applied widely, patient-derived xenografts (PDXs), are regarded to better recapitulate the morphology and heterogeneity of primary tumor tissues, and better predict drug efficacy [8, 9]. PDX models, cancer-bearing mouse models generated by implanting patient’s tumor tissues directly into immunodeficient mice, have the potential to assess personalized therapies [10, 11]. That being said, establishing PDXs is time-consuming and expensive, and engraftment rates vary remarkably across different cancer types, even across different studies for the same cancer type [12–19]. Moreover, large-scale PDX libraries established so far are mainly in developed countries/regions including Europe, the United States, and Japan [20–23]. In China, there is still a lack of large-scale PDX library from gastrointestinal cancers, especially for esophageal squamous cell carcinoma (ESCC), a characteristic tumor type in this country.
In the current study, we obtained fresh surgical specimens or endoscopic biopsy specimens of tumor tissues from patients with gastrointestinal cancers, including ESCC, esophagogastric junction adenocarcinoma (EGJAC), gastric adenocarcinoma (GAC), colorectal cancer, primary liver cancer, metastatic liver cancer from colorectum and pancreas, pancreatic cancer and cholangiocarcinoma. With the tumor tissues taken from 1001 cases, we established 208 PDXs between January 2013 and August 2015. Meanwhile, we assessed engraftment rates, latency period of xenograft formation, association between xenografts and patients’ clinicopathological characteristics and survival for ESCC, EGJAC and GAC.
Methods
Ethical statement
The protocol of this study was approved by the Medical Ethics Committee of Peking University Cancer Hospital, and written informed consent was obtained from each included patient. This study was performed according to principles of the Declaration of Helsinki.
Patients and tissue samples
From January 2013 to August 2015, 1001 specimens of gastrointestinal cancers from 1001 patients were collected, including surgical or endoscopic biopsy specimens from 386 ESCC cases, 183 EGJAC cases, 266 GAC cases and 68 colorectal cancer cases, and surgical specimens from 35 cases of primary liver cancer, 22 cases of metastatic liver cancer from colorectum and pancreas, 33 cases of pancreatic cancer and 8 cases of cholangiocarcinoma. Those specimens were obtained from patients who had undergone tumor resection or endoscopy at the Peking University Cancer Hospital, except for surgical specimens from 63 ESCC cases and 25 EGJAC cases undergone tumor resection at the Anyang Cancer Hospital (Henan, China). Baseline characteristics and clinicopathological data and survival data of the patients were collected, including age, gender, smoking history, drinking history, family history of cancer, the use of chemoradiotherapy before sampling, tumor location, differentiation, tumor-node-metastasis (TNM) stage, and Lauren classification. The follow-up period was ended in January 2021.
Establishment of PDX models
Five- to six-week-old female NOD/SCID mice (Beijing Vital River Laboratory Animal Technology Co., Ltd) were used for all the experiments. Animal experiments were performed in compliance with the Guide for the Care and Use of Laboratory Animals of the NIH.
Fresh surgical or endoscopic biopsy specimens (Passage 0, P0) were immediately soaked in saline after sampling, and were cut with sterilized scissors. The specimens were minced into fragments < 1 mm3 in RPMI 1640 medium supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin, and mixed with equal amount of Matrigel matrix (BD company). The mixture was subcutaneously injected into the flank of each mouse under sterile conditions in the specific pathogen-free facility at Peking University Cancer Hospital.
The mice were weekly monitored for tumor growth. When the tumor became palpable, tumor volume was measured by Vernier calipers twice a week. Once the volume of the subcutaneous tumor reached 200–1500 mm3, or the mice were in poor status characterized by curled and thin furs and emaciation even though the tumor volume was < 200 mm3, mice were sacrificed by inhalation of anesthetics with CO2, and the xenograft tumor tissues were harvested and defined as passage 1 (P1). The xenograft tumor tissues were subsequently re-implanted into NOD/SCID mice, and the tumor tissues harvested after the re-implantation were defined as passage 2 (P2).
In addition, xenograft tumor tissues of P1 and P2 were cryopreserved in suspension for re-implantation, snap-frozen in liquid nitrogen for subsequent extraction of nucleic acid and protein, and fixed in neutral formalin buffer for histopathology.
H&E staining and immunohistochemical staining
The formalin-fixed xenograft tissues of P1 and P2 were embedded in paraffin and sliced into 5 μm-thick sections, which were stained using hematoxylin-eosin (H&E) staining and immunohistochemical (IHC) staining with anti-CK, CD45, CD3, and CD20 antibodies (ZSGB-BIO, Beijing, China) to evaluate the histopathology and exclude tumors suspected as lymphoma. Results were reviewed independently by two pathologists.
Statistical analysis
The associations between clinicopathological characteristics and xenografts were evaluated using multivariable logistic regression adjusted for age and gender. Overall survival (OS) was analyzed using the Kaplan-Meier method and compared using the log-rank test. Cox proportional hazards regression models were used to assess the association of engraftment with overall survival. All tests were two-sided, and a P value < 0.05 was considered statistically significant. All statistical analyses were performed with R (version 4.0.5).
Results
The engraftment rate of PDX models
In the current study, 1001 surgical or endoscopic biopsy specimens from gastrointestinal cancers were implanted between January 2013 and August 2015. Through H&E staining and IHC staining targeted for CK, CD45, CD3, and CD20 on the paraffin-embedded sections of P1 and P2 xenograft tumor tissues, tumors with lymphoma outgrowth were excluded. Finally, a total of 208 PDX models (20.8%, 208/1001) were established, including 82 models of ESCC (21.2%, 82/386), 31 models of EGJAC (16.9%, 31/183), 29 models of GAC (10.9%, 29/266), and 31 models of colorectal cancer (45.6%, 31/68) from surgical or biopsy tissues, and 3 models of primary liver cancer (8.6%, 3/35), 16 models of metastatic liver cancer from colorectum and pancreas (72.7%, 16/22), 14 models of pancreatic cancer (42.4%, 14/33), and 2 models of cholangiocarcinoma (25.0%, 2/8) from surgical tissues (Fig. 1).
Fig. 1.
The engraftment rates of PDX models from gastrointestinal cancers. The numbers in parentheses indicate the number of successful engraftment over the total number
In subsequent analysis, we included only the cancer types with more than 100 specimens available in this study, namely, ESCC (386 specimens), EGJAC (183 specimens), and GAC (266 specimens). Thus, we mainly focused on the engraftment rates, latency period of xenografts, clinicopathological characteristics and survival association with engraftment for ESCC, EGJAC and GAC.
Clinicopathological features of ESCC, EGJAC and GAC patients
The patients’ clinicopathological characteristics are shown in Table 1. Of the patients with ESCC, EGJAC, and GAC, the mean ages (standard deviations) were 61(8), 64(8) and 60(10) years, respectively; most were male, accounting for 79.5%, 84.7%, and 77.1%, respectively; and the majority did not receive chemoradiotherapy before sampling, accounting for 86.8%, 94.0%, and 97.4%, respectively. The specimens of ESCC, EGJAC and GAC were mostly collected at biopsy, accounting for 61.1%, 68.3%, and 74.1%, respectively. ESCC mainly located in the middle esophagus (53.6%), and GAC mainly located in the antrum (66.5%). Well-to-moderate differentiation accounted for 70.5% of all the ESCC cases, while poor differentiation accounted for 61.7% and 74.1% of all the EGJAC and GAC cases, respectively. Over half of the ESCC, EGJAC, and GAC patients were at TNM stage III-IV (56.7%, 76.0%, and 63.2%, respectively). EGJAC and GAC patients were mainly intestinal type (55.7% and 43.6%, respectively) on the basis of Lauren classification. Due to the relatively simplified information collection in patients who only undergone endoscopic examination, approximately 12.2-22.2% of the information on smoking and drinking history and family history of cancer were missing.
Table 1.
Clinicopathological characteristics of ESCC, EGJAC and GAC patients
| ESCC (n = 386) |
EGJAC (n = 183) |
GAC (n = 266) |
||
|---|---|---|---|---|
| Age, mean(SD), yrs | 61 (8) | 64 (8) | 60 (10) | |
| Gender, No. (%) | ||||
| Male | 307 (79.5) | 155 (84.7) | 205 (77.1) | |
| Female | 79 (20.5) | 28 (15.3) | 61 (22.9) | |
| Chemoradiotherapy before sampling, No. (%) | ||||
| No | 335 (86.8) | 172 (94.0) | 259 (97.4) | |
| Yes | 51 (13.2) | 11 (6.0) | 7 (2.6) | |
| Specimen Type, No. (%) | ||||
| Biopsy | 236 (61.1) | 125 (68.3) | 197 (74.1) | |
| Surgery | 150 (38.9) | 58 (31.7) | 69 (25.9) | |
| Tumor location, No. (%) | ||||
| Upper | 46 (11.9) | Fundus/Body | 89 (33.5) | |
| Middle | 207 (53.6) | Antrum | 177 (66.5) | |
| Lower | 133 (34.5) | |||
| Differentiation, No. (%) | ||||
| Well/Moderate | 272 (70.5) | 70 (38.3) | 68 (25.6) | |
| Poor | 112 (29.0) | 113 (61.7) | 197 (74.1) | |
| Missing | 2 (0.5) | 1 (0.3) | ||
| TNM stage, No. (%) | ||||
| I/II | 135 (35.0) | 34 (18.6) | 82 (30.8) | |
| III/IV | 219 (56.7) | 139 (76.0) | 168 (63.2) | |
| Missing | 32 (8.3) | 10 (5.4) | 16 (6.0) | |
| Lauren classification, No. (%) | ||||
| Diffuse | 21 (11.5) | 49 (18.4) | ||
| Intestinal | 102 (55.7) | 116 (43.6) | ||
| Mixed | 29 (15.8) | 90 (33.8) | ||
| Missing | 31 (17.0) | 11 (4.2) | ||
| Smoking history, No. (%) | ||||
| Never | 120 (31.1) | 78 (42.6) | 120 (45.1) | |
| Current/Ex − smoker | 210 (54.4) | 66 (36.1) | 87 (32.7) | |
| Missing | 56 (14.5) | 39 (21.3) | 59 (22.2) | |
| Drinking history, No. (%) | ||||
| Never | 152 (39.4) | 101 (55.2) | 153 (57.5) | |
| Current/Ex − drinker | 176 (45.6) | 43 (23.5) | 54 (20.3) | |
| Missing | 58 (15.0) | 39 (21.3) | 59 (22.2) | |
| Family history of cancer, No. (%) | ||||
| No | 240 (62.2) | 103 (56.3) | 158 (59.4) | |
| Other cancers | 36 (9.3) | 31 (16.9) | 37 (13.9) | |
| Same cancer | 63 (16.3) | 11 (6.0) | 17 (6.4) | |
| Missing | 47 (12.2) | 38 (20.8) | 54 (20.3) | |
ESCC Esophageal squamous cell carcinoma, EGJAC Esophagogastric junction adenocarcinoma, GAC Gastric adenocarcinoma
Factors associated with engraftment for ESCC, EGJAC and GAC
The engraftment success rates of ESCC, EGJAC and GAC were 21.2% (82/386), 16.9% (31/183) and 10.9% (29/266), respectively (Fig. 2A). For ESCC, EGJAC, and GAC, the average latency period of xenograft formation was 76.2, 90.5, and 85.2 days from P0 to P1 respectively, which decreased to 52.5, 54.8, and 52.6 days from P1 to P2 respectively (Fig. 2B). H&E staining showed that the xenografts retained the histopathology of the corresponding primary tumor tissues (Fig. 2C)
Fig. 2.
Success rates of engraftment, latency period and histopathology of xenografts for ESCC, EGJAC and GAC. A Bar chart depicting the success rates of PDX models for ESCC, EGJAC, and GAC (q values were adjusted by false discovery rate using Bonferroni correction). B Time to passage by cancer type (*** p < 0.001). C Histology of primary tumors and xenografts. Scale bars represent 20 μm. ESCC Esophageal squamous cell carcinoma, EGJAC Esophagogastric junction adenocarcinoma, GAC gastric adenocarcinoma, NS not significant
To better control for confounding factors, we conducted multivariable analysis to explore patients’ clinicopathological features associated with engraftment for ESCC, EGJAC and GAC, including age, gender, specimen type (surgery versus endoscopic biopsy), differentiation, treatment status before sampling, TNM stage (I/II versus III/IV), tumor location, Lauren’s classification, smoking history, drinking history, and family history of cancer (Table 2).
Table 2.
Factors associated with engraftment for ESCC, EGJAC and GAC
| ESCC | EGJAC | GAC | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Engraftment success no./total no.(%) | Adjusted OR (95% CI) | p value | Engraftment success no./total no.(%) | Adjusted OR (95% CI) | p value | Engraftment success no./total no.(%) | Adjusted OR (95% CI) | p value | ||
| Age, yrs | ||||||||||
| < 60 | 40/159 (25.2) | Ref. | 9/47 (19.1) | Ref. | 7/117 (6.0) | Ref. | ||||
| ≥ 60 | 42/227 (18.5) | 0.78(0.47–1.29) | 0.332 | 22/136 (16.2) | 0.83 (0.36–2.04) | 0.668 | 22/149 (14.8) | 2.72 (1.17–7.10) | 0.028 | |
| Gender | ||||||||||
| Male | 74/307 (24.1) | Ref. | 28/155 (18.1) | Ref. | 20/205 (9.8) | Ref. | ||||
| Female | 8/79 (10.1) | 0.38 (0.16–0.80) | 0.016 | 3/28 (10.7) | 0.55 (0.13–1.71) | 0.354 | 9/61 (14.8) | 1.59 (0.65–3.65) | 0.287 | |
| Specimen Type | ||||||||||
| Biopsy | 34/236 (14.4) | Ref. | 19/125 (15.2) | Ref. | 16/197 (8.1) | Ref. | ||||
| Surgery | 48/150 (32.0) | 3.65 (2.15–6.18) | < 0.001 | 12/58 (20.7) | 1.53 (0.68–3.44) | 0.304 | 13/69 (18.8) | 2.34 (1.05–5.22) | 0.039 | |
| Differentiation | ||||||||||
| Well/Moderate | 66/272 (24.3) | Ref. | 14/70 (20.0) | Ref. | 12/68 (17.6) | Ref. | ||||
| Poor | 16/112 (14.3) | 0.52 (0.28–0.95) | 0.033 | 17/113 (15.0) | 0.47 (0.04–5.83) | 0.555 | 17/197 (8.6) | 0.4 (0.18–0.92) | 0.030 | |
| Lauren classification | ||||||||||
| Diffuse | 2/21 (9.5) | Ref. | 2/49 (4.1) | Ref. | ||||||
| Intestinal | 18/102 (17.6) | 1.96 (0.42–9.30) | 0.395 | 18/116 (15.5) | 4.92 (1.06–22.91) | 0.043 | ||||
| Mixed | 3/29 (10.3) | 1.06 (0.16–7.06) | 0.948 | 8/90 (8.9) | 2.67 (0.53–13.5) | 0.234 | ||||
| Chemoradiotherapy before sampling | ||||||||||
| No | 67/335 (20.0) | Ref. | 28/172 (16.3) | Ref. | 29/259 (11.2) | Ref. | ||||
| Yes | 15/51 (29.4) | 1.68 (0.86–3.29) | 0.127 | 3/11 (27.3) | 1.86 (0.46–7.51) | 0.381 | 0/7 (0.0) | NA | ||
| TNM stage | ||||||||||
| I/II | 27/135 (20.0) | Ref. | 5/34 (14.7) | Ref. | 6/82 (7.3) | Ref. | ||||
| III/IV | 48/219 (21.9) | 1.03 (0.60–1.76) | 0.917 | 25/139 (18.0) | 1.21 (0.42–3.47) | 0.717 | 22/168 (13.1) | 1.91 (0.74–4.98) | 0.183 | |
| Tumor location | ||||||||||
| Upper | 6/46 (13.0) | Ref. | Fundus/Body | 8/89 (9.0) | Ref. | |||||
| Middle | 43/207 (20.8) | 1.50 (0.59–3.82) | 0.398 | Antrum | 21/177 (11.9) | 1.3 (0.55–3.10) | 0.550 | |||
| Lower | 33/133 (24.8) | 1.74 (0.66–4.57) | 0.261 | |||||||
| Smoking history | ||||||||||
| Never | 20/120 (16.7) | Ref. | 11/78 (14.1) | Ref. | 15/120 (12.5) | Ref. | ||||
| Current/Ex − smoker | 52/210 (24.8) | 1.25 (0.63–2.49) | 0.518 | 12/66 (18.2) | 1.15 (0.45–2.95) | 0.767 | 8/87 (9.2) | 0.85 (0.31–2.38) | 0.762 | |
| Drinking history | ||||||||||
| Never | 28/152 (18.4) | Ref. | 16/101 (15.8) | Ref. | 21/153 (13.7) | Ref. | ||||
| Current/Ex − drinker | 41/176 (23.3) | 1.03 (0.56–1.89) | 0.922 | 7/43 (16.3) | 0.87 (0.32–2.37) | 0.782 | 2/54 (3.7) | 0.24 (0.05–1.12) | 0.070 | |
| Family history of cancer | ||||||||||
| No | 58/240 (24.2) | Ref. | 19/103 (18.4) | Ref. | 21/158 (13.3) | Ref. | ||||
| Other cancers | 6/36 (16.7) | 0.66 (0.26–1.67) | 0.379 | 3/31 (9.7) | 0.47 (0.13–1.70) | 0.248 | 3/37 (8.1) | 0.56 (0.16–2.01) | 0.374 | |
| Same cancer | 8/63 (12.7) | 0.50 (0.22–1.12) | 0.091 | 2/11 (18.2) | 0.88 (0.17–4.47) | 0.880 | 0/17 (0.0) | NA | 0.992 | |
OR Odds ratio, Ref Reference, ESCC Esophageal squamous cell carcinoma, EGJAC Esophagogastric junction adenocarcinoma, GAC Gastric adenocarcinoma, NA Not applicable
For ESCC and EGJAC, engraftment rates of tumor tissues from patients younger than 60 years old were slightly higher than the rates of those from patients over 60 years old (ESCC: 25.2% [40/159] versus 18.5% [42/227], EGJAC: 19.1% [9/47] versus 16.2% [22/136]), but with no statistical significance. For GAC, in contrast, engraftment rate in the over 60 years age group (14.8%, 22/149) was significantly higher than that in the younger than 60 years age group (6.0%, 7/117) (adjusted OR = 2.72, 95% CI = 1.17–7.10, P = 0.028).
For ESCC, engraftment rate of tissues from males (24.1%, 74/307) was significantly higher than that from females (10.1%, 8/79) (adjusted OR = 0.38, 95% CI = 0.16–0.80, P = 0.016); similar gender difference was also observed for EGJAC (18.1% [28/155] versus 10.7% [3/28]). For GAC, in contrast, engraftment rate of tissue specimens from males (9.8%, 20/205) was lower than that from females (14.8%, 9/61).
In terms of specimen type, engraftment rate of surgical specimens was significantly higher than that of biopsy specimens for ESCC (32.0% [48/150) versus 14.4% [34/236], adjusted OR = 3.65, 95% CI = 2.15–6.18, P < 0.001) and GAC (18.8% [13/69] versus 8.1% [16/197], adjusted OR = 2.34, 95% CI = 1.05–5.22, P = 0.039), also for EGJAC but with no statistical difference (20.7% [12/58] versus 15.2% [19/125], adjusted OR = 1.53, 95% CI = 0.68–3.44, P = 0.304).
Well-to-moderate differentiation was found associated with significantly higher engraftment rate compared with poor differentiation for ESCC (24.3% [66/272] versus 14.3% [16/112], adjusted OR = 0.52, 95% CI = 0.28–0.95, P = 0.033) and GAC (17.6% [12/68] versus 8.6% [17/197], adjusted OR = 0.4, 95% CI = 0.18–0.92, P = 0.030), and associated with slightly higher engraftment rate for EGJAC (20.0% [14/70] versus 15.0% [17/113], adjusted OR = 0.47, 95% CI = 0.04–5.83, P = 0.555).
According to Lauren classification, for EGJAC, engraftment rate was higher for tissues classified as intestinal type (17.6%, 18/102) compared with diffuse type (9.5%, 2/21) and mixed type (10.3%, 3/29); engraftment rate was statistically significantly higher for GAC tissues classified as intestinal type than for tissues of diffuse type (15.5% [18/116] versus 4.1% [2/49], adjusted OR = 4.92, 95% CI = 1.06–22.91, P = 0.043).
No statistically significant difference was observed in engraftment rates across the strata by treatment status before sampling, TNM stage (I/II versus III/IV), tumor location, smoking history, drinking history, and family history of cancer for ESCC, EGJAC and GAC.
Survival by engraftment for ESCC, EGJAC and GAC
Up to the end of follow-up in January 2021, the median follow-up duration for ESCC, EGJAC and GAC patients was 46, 64, and 64 months, respectively. We used Kaplan-Meier method to investigate the association between patients’ survival and PDXs. As shown in Fig. 3, for ESCC, the survival time of patients with successful engraftment was similar to that without xenograft formation (adjusted HR = 0.83, P = 0.364). For EGJAC, the survival of patients with successful engraftment was worse than that of those without xenograft formation, but the difference was not statistically significant (adjusted HR = 1.78, P = 0.126). For GAC, the survival of patients with successful engraftment was statistically significantly worse compared with those without xenograft formation (adjusted HR = 2.28, P = 0.015).
Fig. 3.
Kaplan–Meier analysis of overall survival of patients and xenografts engraftment status. For ESCC, the hazard ratio (HR) was adjusted for age, gender, smoke status, alcohol using, TNM stage, family history and differentiation. For EGJAC and GAC, HRs were adjusted for age, gender, smoking, alcohol using, TNM stage, family history, differentiation and Lauren classification. ESCC Esophageal squamous cell carcinoma, EGJAC Esophagogastric junction adenocarcinoma, GAC gastric adenocarcinoma, NS not significant, NA not applicable
Discussion
PDXs retain key histopathological and biological characteristics of the original tumors and are therefore regarded as the most reliable model for drug development in preclinical phase and individualized therapy [9–11].
This work was the first on establishment of large-scale Chinese PDX models from gastrointestinal cancers, including ESCC, EGJAC, GAC, colorectal cancer, primary liver cancer, metastatic liver cancer from colorectum and pancreas, pancreatic cancer and cholangiocarcinoma. A total of 1001 fresh surgical or endoscopic biopsy specimens were implanted between 2013 and 2015, from which 208 PDXs were successfully established, resulting in an overall engraftment rate of 20.8% (208/1001). Metastatic liver cancer from colorectum and pancreas (72.7%, 16/22), colorectal cancer (45.6%, 31/68), and pancreatic cancer (42.4%, 14/33) had high engraftment rates, similar to previous reports [24–26], supporting the reliability of our procedure of engraftment.
There were three cancer types with more than 100 specimens for implantation, including ESCC (n = 386), EGJAC (n = 183), and GAC (n = 266), thus, we focused our analysis on these three types of upper gastrointestinal cancers. Engraftment rate, latency period of xenografts, factors associated with and survival status by engraftment for ESCC, EGJAC and GAC were assessed.
The success rates of engraftment for ESCC, EGJAC and GAC (21.2% [82/386], 16.9% [31/183], and 10.9% [29/266], respectively) were relatively lower than the rates previously reported. Specifically, the tumor engraftment rates of PDX models reported in previous studies were 13.3–55.5% in ESCC [12, 16, 17], 26% in EGJAC [19], and 15–28% in GAC [15, 18], respectively. This may be mainly due to the type of tissues transplanted. In the current study, endoscopic biopsy tissues dominated, in which only a small volume of tissues were available for implantation. The latency period of xenografts was approximately 80 days for P1 and 50 days for P2 in our current study, regardless of the cancer types. For all the three cancers (ESCC, EGJAC, and GAC), we observed a significant reduction in latency period of xenografts from P1 to P2, similar to previous reports [23], which may be explained by the tumor adaptation to the mice’s environment.
In this study, we found that for ESCC, male patients, surgical tissues, and tumors with well-to-moderate differentiation had a higher success rate of PDXs; and for GAC, patients ≥ 60 years, surgical tissues, tumors with well-to-moderate differentiation, and tumors of intestinal type had a higher success rate of PDXs. Similar as in ESCC, tumor tissues from younger (< 60 years) and male EGJAC patients achieved higher engraftment rates, but the age and gender differences in EGJAC were not statistically significant. In addition, we analyzed the association between survival of patients and successful PDX engraftment. No association was found for ESCC. For EGJAC and GAC, patients with successful engraftment had poorer survival, and the survival difference was statistically significant for GAC. To sum up, although ESCC, EGJAC and GAC are all upper gastrointestinal cancers, their engraftment rates, factors associated with and survival status by engraftment varied, which may be due to their differences in cell type, pathogenesis and etiology. Exploration of the mechanism underlying successful engraftment is warranted.
Cancer cell lines in vitro or cell line-derived xenograft models have long been used for drug development in preclinical stage, yet they are inferior to in vivo in terms of cells or tissues phenotype and inevitably affected by contamination among cell lines after many passages [27, 28]. In contrast, PDX models, which have been increasingly used in recent years, preserve most of the tumor characteristics and are regarded to be more reliable in predicting drug efficacy. With the rapid development of next-generation sequencing technology, PDX models as an ‘avatar’ of a patient are genotyped for guiding personalized therapy and exploring the underlying mechanisms of drug resistance.
Current PDX models still have shortages in some respects, such as the lack of immune cells compared to the tumor microenvironment in human body, low success rate of engraftment, and long latency period of xenografts. Relevant techniques need to be improved for application in cancer research.
Some limitations of this study are to be considered. First, endoscopic biopsy specimens were smaller in volume than surgical specimens, thus, it was more difficult to keep the corresponding primary tumor tissues when establishing PDX models using endoscopic biopsy specimens. Second, a few patients only undergone endoscopic examination and did not receive further treatment in the study hospital, for whom clinicopathological data and follow-up information were not available.
Conclusions
In summary, we successfully established a Chinese library of 208 PDXs from gastrointestinal cancers, especially ESCC, the predominant histological type of esophageal cancers in China. We are further conducing genomic and functional studies on those PDX models, which will facilitate understanding of cancer mechanisms and shedding light on potential novel therapeutics.
Acknowledgements
We thank all the patients who consented providing their tumor specimens and all the endoscopists and sugeons who performed endoscopic/surgical specimen collection for this study.
Abbreviations
- PDX
Patient-derived xenografts
- ESCC
Esophageal squamous cell carcinoma
- EGJAC
Esophagogastric junction adenocarcinoma
- GAC
Gastric adenocarcinoma
- TNM
Tumor-node-metastasis
Authors’ contributions
YK conceived and designed the study. QW, JJ, LS, CH, RX, KC, NW, CC, YH, LZ contributed to the clinical specimens. YL, HC, ZH, YP, WY, JL, ML, ZL, and FL carried out the experimental work. YL, WH, BD, ZH, and HC performed data collections and analysis. YL, WH, and YK drafted the manuscript. All authors reviewed the manuscript.
Funding
This work was supported by grants from National Natural Science Foundation of China (81903155), Beijing Municipal Natural Science Foundation (7202023), Beijing Municipal Science and Technology Commission (Z141100002114046), and 973 Project of National Ministry of Science and Technology (2012CB910800).
Data availability
No datasets were generated or analysed during the current study.
Declarations
Ethics approval and consent to participate
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All animal experiments were performed in accordance with and approved by the animal experimental guidelines of Peking University Cancer Hospital and followed internationally recognized ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. The study was conducted in accordance with the principles expressed in the Declaration of Helsinki. The study was approved and supervised by the research ethics committee of Peking University Cancer Hospital & Institute, Beijing, China (No. 2013011516).
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Ying Liu, Wei He and Qi Wu contributed equally to this work.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
No datasets were generated or analysed during the current study.