Establishment and characterization of patient-derived xenografts for hormone-naïve and castrate-resistant prostate cancers to improve treatment modality evaluation

Characteristics of the PDX models

Fresh surgical PC specimens, from a patient with CRPC (B45354) and from another with HNPC (D17225) were used to establish two PC PDX models with differing androgen sensitivities. The tumor specimens were implanted into the renal capsule of male immune-compromised NPG/Vst (NOD-Prkdc scid Il2rgnull) mice and supplemented with testosterone. Initial tumor growth was measured with a near-infrared fluorescence (NIRF) optical imaging system twice weekly using the NIRF heptamethine carbocyanine dye MHI-148. The dye was seen to have preferential uptake in tumor cells but not in normal cells. This can be employed directly for non-invasive dual imaging and for the targeting of agents in human cancers without the need of chemical conjugation [20, 21]. As shown in Figure 1A (top), NIRF signals with strong intensity were obtained in the kidney 3 months after the transplantation of D17225 or B453543 specimens, indicating tumor formation. After euthanizing the mice, tumor nodules were observed (Figure 1A, bottom). Tumors that grew were dissected from the host mice and further passaged subcutaneously into new NPG/Vst male mice. The initial PDX model was established within 3–6 months. We considered three passages to indicate of an established PDX line.

Figure 1. Establishment of molecular heterogeneous PDX models with maintained pathological features of patient tumors. (A) NIRF optical imaging of NPG mice with PC xenograft tumors using MHI-148 dye (Top). The PDX models of PC were established by implantation of clinical tumor specimens D17225 and B45353 into the renal capsule of NPG mice. Gross morphology of PC tissue-implanted mouse kidney (bottom). (B, C) H&E and IHC analyses of tumor tissues derived from both PDX models and patient samples (D17225 and B45353). (D) Multiple organ metastasis PDX models from B45354 (CRPC patient). H&E staining of metastatic tumors (including the lung and liver). Original magnification, 400×; scale bars represent 20 μm.

Fidelity of the PDX models to the primary tumor

Both the D17225 and B45354 PDX models exhibited diverse histological features (Table 1). Hematoxylin and eosin (H&E) staining confirmed that the PDX tumor tissues exhibited identical morphological characteristics to that of the original tumor. In order to distinguish between human and mice tissues, the human mitochondria was used as a marker, and was strongly expressed in both human xenograft tissues (Supplementary Figure 1). In the D17225 PDX, the tumor basal cells disappeared and a large number of irregular sieve-like structures appeared. The cancer cells were enlarged and infiltrated the surrounding space. Castration-resistant B45354 PDX tumors retained the glandular structure of the primary tumor with relatively clear boundaries. In the D172225 PDX, both AR- and prostate-specific antigen (PSA)-specific immunohistochemistry (IHC) profiles were generally maintained (Figure 1B and 1C). Additionally, the expression levels of the neuroendocrine carcinoma markers synaptophysin (SYP) and chromogranin A (CGA) were concordant with those in the original tissue. However, the expression of most of the proteins in the B45354 patient were only weakly positive, whereas that in the B45354 PDX mice were negative; only CGA was weakly expressed in the PDX model. Moreover, high metastatic potential was observed in the B45354 PDX model, partly because of greater vascularization at the graft site, and multi-organ metastases ultimately developed, including in the lung and liver (Figure 1D). The tumor cells invaded the right seminal vesicles and bladder tissue, resulting in a higher degree of malignancy than that observed in the patient. The tumor of castration-resistant B45354 reached a maximum volume of 800 mm³ 4 weeks after implantation and grew faster than the androgen-dependent D17225 tumor.

Factors influencing establishment of the PC PDX models

To examine the main factors influencing the establishment of the PC PDX models, tumor tissue from the D17225 PDX model was implanted into mice. The control group was only subcutaneously implanted with the tissue, whereas in the testosterone groups implantation was performed subcutaneously (testosterone 1) or under the renal capsule (testosterone 2) and testosterone was supplemented. The serum testosterone levels in androgen-implanted mice were measured weekly. After one week, the testosterone levels reached a peak and then gradually decreased but remained between 20–60 ng/dl (Figure 2A), significantly higher than that of normal male mice (6.0-7.8 ng/dl). Mice supplemented with androgen displayed faster tumor growth than those in the control group (Figure 2B). The renal capsule transplantation group exhibited significantly shortened growth latency (from the first day of transplantation to the time of tumor growth to 100 mm³) (Figure 2C).

Figure 2. Characteristics of the hormone-naïve D17225 PDX model. (A–C) Factors influencing the establishment of the PDX model. (A) Change in testosterone levels in androgen-implanted mice. (B) Effect of supplementation with androgen on PC tumor growth. (C) Effect of supplementation with androgen on the growth latency of the PC tumor. Results are shown as the means ± SD (n = 5). (D–E) Induction of CRPC in the D17225 PDX model. (D) Tumor volumes of D17225 xenografts and mouse serum PSA levels at various time points before, during, and after castration-induced CRPC development. (E) H&E staining of D17225 tumor sections, and the levels of AR and PSA in tumor sections (IHC analysis) at various time points before and after host castration and in recurrent tumors. The scale bar indicates 100 mm. Cx indicates castration weeks.

Induction of CRPC

One week after the castration of mice bearing D17225 tumors, the tumor volume was markedly reduced, accompanied by a substantial decrease in host serum PSA levels (Figure 2D). This mimics the clinical response of patients with PC to ADT. A few months after castration, the host serum PSA levels increased (Figure 2D). One week after host castration, tumor growth slowed, and AR expression markedly decreased, indicating reduced AR transcriptional activity. However, after tumor recurrence, these markers were positively expressed at higher levels (Figure 2E).

Short tandem repeat (STR) profile and AR mutation

To ensure the originality and maintenance of the genotypes of the PDX models, we performed STR analyses, and demonstrated distinct STRs in each model. The 16 STR loci of tumor tissues derived from PDX models were confirmed to be identical to the loci from the patient tumor tissue (Table 2). Further analysis demonstrated the loss of JARID1D expression in the tumor tissue from the B45354 PDX, which was similar to the pattern observed in the PC cell line, PC-3 (Figure 3A). This gene—located on the Y chromosome—is a prognostic marker and suppressor of prostatic tumor invasion and metastasis [22, 23]. Polymerase chain reaction (PCR) from B45354 barely amplified the specific AR band, whereas D17225 showed an amplified band and the fragment size was consistent with that of the AR target fragment after sequencing. The expression of AR, as determined by reverse transcription (RT)-PCR analysis was consistent with the results of IHC. In addition, although AR expression was nearly negative in B45354 tumor tissues, an increase in the levels of the androgen receptor variant 7 (ARV7) was detected in the PDX tissue by RT-PCR, but weak positive expression was detected by IHC (Figure 3B and 3C).

Figure 3. Expression of AR and JARID1D by RT-PCR analysis. (A) Expression of JARID1D in tumors tissue of different PDX models and PC cells were detected by RT-PCR. (B) Expression of AR and ARV7 in tumors tissues of different PDX models detected by RT-PCR. Data are shown as the means ± SD of three independent experiments performed in triplicate. (C) Expression of ARV7 in tumors tissues of B45354 PDX models detected by IHC. Original magnification, 400×; scale bars represent 20 μm.

Responses to castration and docetaxel

Because ADT is the first-line therapy for advanced PC and docetaxel was the first therapeutic agent shown to extend survival in men with metastatic CRPC [3], we evaluated the responses of different PDX models to castration, docetaxel and combination of the two. The therapeutic effect was assessed by examining changes in the tumor volume and body weight of tumor-bearing mice. As shown in Figure 4A, in the D17225 PDX model, docetaxel (Dctx), Castration (Cas) or the combined castration + docetaxel (Dctx+Cas) treatment significantly inhibited tumor growth (P < 0.01 compared to controls). In the B45354 PDX model, although docetaxel also inhibited tumor growth, there was no significant difference with respect to the control group, and both castration + docetaxel and docetaxel treatments resulted in significant loss in the body weight of the mice (Figure 4B).

Figure 4. Responses to castration and docetaxel in PDX models. Change in the (A) tumor volume and (B) weight of PDX models after treatment at various time points. (C) Serum tPSA levels in mice bearing PDX tumors after treatment for 30 days. **P < 0.01 compared to the control.

The serum total prostate specific antigen (tPSA) level is an important indicator used in the clinical diagnosis and treatment evaluation of PC [1]. In the D17225 model, tPSA levels in all tumor-bearing mice were above 100 ng/mL. The tPSA levels were the lowest in the docetaxel + castration group (average 2.58 ng/mL), followed by the castration group (average, 3.65 ng/mL). In contrast, the average tPSA content in the docetaxel group was 30.92 ng/mL (Figure 4C). In the B45354 model, the serum tPSA level was below the critical minimum in all mice, which was consistent with the results of histopathological analysis.

We also evaluated the therapeutic effect by observing the changes in histological morphology with H&E staining (Figure 5). In the D17225 model, the tumor cells were abundant and varied in size, and the morphology of the nucleus was variable. After castration or docetaxel treatment, the number of tumor cells decreased and the matrix component increased. After castration + docetaxel treatment, the number of tumor cells decreased gradually and did not exhibit typical characteristics (Figure 5A). In the B45354 model, the different treatments did not alter the structure of the tumor tissue significantly (Figure 5B). The model even displayed liver metastasis after treatment with docetaxel (Figure 5C) with metastatic percentage reaching 30%. Moreover, the treatment of B45354 PDXs with castration + docetaxel induced the expression of the neuroendocrine markers CGA and CYP at high levels, thus displaying a feature of neuroendocrine carcinoma (Figure 5D).

Figure 5. H&E staining and IHC analysis of different treatment groups. Results of H&E staining and IHC analysis in the different treatment groups in the (A) D17225 and (B) B45354 PDX model. (C) Representative images of liver metastasis and the parental tumors after treatment with docetaxel in the B45354 PDX model. (D) Expression of neuroendocrine carcinoma markers SYP and CGA after treatment with castration + docetaxel in the B45354 PDX model.

Animals

Six to seven-week-old male NPG/Vst (NOD-Prkdc scid Il2rg null) mice were purchased from Beijing Vitalstar Biotechnology (Beijing, China) and housed in a pathogen-free environment at the Laboratory Animal Center of the Fourth Military Medical University (FMMU, Xi’an, China). The mice were anesthetized with ketamine (100 mg/mL, i.p.) and xylazine (20 mg/mL, i.p.) and maintained under isoflurane during surgery and imaging. All animal experiments were approved by the Institutional Animal Care and Use Committee of FMMU (Protocol No. 16013).

Cells and tumor specimens

Both human prostate cancer cell lines LNCaP and PC3 were purchased from the American Type Culture Collection (ATCC, Manassas, VA). Fresh prostate cancer specimens were obtained from the Xijing Hospital of the FMMU, and informed consent was obtained from both patients. One specimen was derived from a clinical HNPC patient (D17225) and the other was derived from a CRPC patient (B45354). The use of these specimens in research was approved by the Medical Eethics Committee of Xijing Hospital (KY20193035). After dissection, the specimens were cut into 1–3-mm³ fragments and the stromal capsules and necrotic tissue were removed.

Establishment of the PDX models

Two to three PC specimen grafts mixed with an appropriate amount of matrigel (BD Biosciences, Franklin Lakes, NJ, USA) were implanted between the capsule and underlying parenchyma of each kidney in the male mice under general anesthesia [31]. To maintain host testosterone levels, implants of testosterone (4 Androsten 17b-01–3-one, Sigma Chemical Co., St. Louis, MO, USA) were inserted intraperitoneally at a dose of 40 mg/capsule per mouse [6]. Plasma testosterone levels in tumor-bearing mice with implants were monitored by enzyme-linked immunosorbent assay (ELISA). Initial tumor growth was measured with an NIRF optical imaging system (Caliper Life Sciences, Hopkinton, MA, USA) twice weekly. The mice were intravenously injected with MHI-148 (1 μmol/kg; provided by Dr. Leland W.K. Chung, Cedars-Sinai Medical Center, Los Angeles, CA, USA), and the NIRF intensity in the regions of interest were measured using a Lumina II Imaging System at 24 h after dye administration. The mice were monitored for up to 6 months post-implantation to examine initial tumor growth [20], and MHI-148 uptake was examined in accordance with the published methods [19]. Tumor samples from PDX models were collected at each stage and frozen or fixed with 4% polyformaldehyde.

STR analysis and PCR amplification

Genomic DNA was extracted from PDX tissues for STR analysis with a tissue genomic DNA extraction kit from Tiangen Biochemical Technology Co., Ltd. (Beijing, China). To evaluate gene expression, total RNA isolated from the PDX tissues was used for RT-PCR as previously described [3, 23]. The primer sequences of the target genes (AR, AR7, JARID1D) are listed in Supplementary Table 1.

Treatment

Male NPG mice were subcutaneously implanted with tumor grafts. When the tumor size exceeded 80–100 mm³, the mice were divided into the following four groups: 1) castration group with surgical removal of the bilateral testes and their attachments, 2) docetaxel group that received docetaxel treatment at a dose of 2 mg/kg via tail vein injection twice weekly, 3) castration + docetaxel group, and 4) control group containing non-castrated male mice. The docetaxel injection solution was procured from Sanofi Pharmaceutical Co., Ltd. (Hangzhou, China). Phosphate-buffered saline (control group) was injected via the tail vein twice weekly. The tumor volume and body weight of the mice in the PDX model were measured once every 3 days. The body weight of tumor-bearing mice and the maximum length (l) and width (w) of the tumor were measured; tumor volume (V) was calculated using the formula V = ½ × (w² × l). After 30 days of treatment, the experiment was terminated, and the blood was collected from the mice to determine the tPSA levels in the serum by ELISA. Then, all tumor-bearing mice were euthanized.

H&E and IHC staining

A section of each tumor was fixed in 4% paraformaldehyde blocks and the morphology was compared to that of the original patient tumor by H&E staining and IHC analysis. The following primary antibodies were used: CGA (1:100; Abcam, Cambridge, UK), SYP (1:50; Santa Cruz), rabbit anti-human anti-PSA, anti-AR monoclonal antibodies (1:100, Abcam, Cambridge, UK). Anti-AR(AR-V7 specific) antibodies (1:200, Abcam, Cambridge, UK) and anti-human mitochondria antibodies (1:100, Abcam, Cambridge, UK). The secondary antibody was a biotin-labeled goat anti-rabbit, which was used according to a published protocol [1].

Statistical analysis

All data are presented as the mean ± SD from at least three independent experiments. The statistical significance of all data was analyzed by a two-tailed unpaired Student’s t test, and a P value < 0.05 was considered statistically significant.