Novel preclinical models, therapies and biomarkers for testicular cancer
Rosas Plaza, Fernanda
DOI:
10.33612/diss.119056452
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Publication date: 2020
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Rosas Plaza, F. (2020). Novel preclinical models, therapies and biomarkers for testicular cancer. Rijksuniversiteit Groningen. https://doi.org/10.33612/diss.119056452
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Chapter 5
AFP levels do not reflect tumor response to cisplatin in
testicular cancer patient-derived xenograft models
Ximena Rosas-Plaza, Gerda de Vries, Gert Jan Meersma, Albert J.H. Suurmeijer, Marcel A. van Vugt, Jourik A. Gietema, and Steven de Jong.LDH (lactate dehydrogenase), AFP (alpha-fetoprotein) and ß-HCG (human chorionic gonadotropin) are used in diagnosis, staging and follow-up of testicular cancer (TC) patients. However, not all testicular tumors secrete these proteins. Moreover, elevated LDH and AFP serum levels can be found with other cancer types different from TC. In this report we aimed to validate patient derived xenografts (PDX) as suitable models to study TC biomarkers. We selected three PDX models derived from both AFP-positive and ß-HCG-positive patients. Two models were derived from cisplatin sensitive TC patients and one from a cisplatin-resistant TC patient. AFP and ß-HCG serum levels were estimated using an ELISA. TC1, TC4 and TC5 all produced AFP. The PDX model derived from a patient, who presented with high ß-HCG serum levels, did not secrete detectable ß-HCG levels in sera of mice. To monitor AFP levels in TC PDX mice during treatment, we took serum samples from mice at start and end of a cisplatin treatment cycle. There was an inverse relation between AFP serum levels and tumor volume or response to treatment in both PDX models. In conclusion, our results suggest that in PDX models from TC AFP and ß-HCG should not be used as response evaluation tool.
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AFP levels do not reflect tumor response to cisplatin in testicular cancer patient-derived xenograft models
Introduction
Testicular cancer has a good prognosis. Cisplatin-based chemo achieves an over 80% 5-year survival in metastatic patients regardless of the tumor histological type. However, patients who develop relapse of refractory disease after first-line
treatment have a much lower survival1, 2. The tumor markers lactate dehydrogenase
(LDH), alpha-fetoprotein (AFP) and human chorionic gonadotropin (ß-HCG) are secreted by testicular cancer (TC) cells and are measurable in sera of TC patients. Serum levels of tumor markers are used for diagnosis, staging and follow-up of TC patients. However, these markers face several limitations. For example, the proportion of patients with elevated tumor markers depends on the tumor histological subtype. Around 90% of non-seminoma tumors will
produce AFP or ß-HCG and 30% of seminomas will secrete ß-HCG3. LDH, the
least specific marker of all three, is elevated in 40-60% of patients regardless of
the histological tumor type4.
Patient Derived Xenograft (PDX) models could be an interesting alternative to study tumor markers in more depth. These models are now more commonly used in cancer research due to the higher resemblance to human tumors than human cell line-based xenografts. Feasibility of PDX models to investigate serum
biomarkers has been demonstrated in pancreatic cancer5 and breast cancer6
showing concordance of clinical features (i. e. tumor presence and response to treatment) with biomarker levels.
Several TC PDX models, either orthotopically or subcutaneously, have been established but TC tumor markers were not investigated. Recently, we developed testicular PDX models by implanting testicular tumors subcutaneously into NOD SCID mice. These PDX models were established from both chemotherapy sensitive and resistant TC patients. We demonstrated that human stroma is
taken over by mouse stroma7 and only human tumor remains, allowing us to
analyze tumor specific products in mouse sera. Assays to determine LDH serum levels are based on the enzymatic activity of LDH, which are neither tumor cell specific nor species-specific. In the present study, we therefore evaluated the possibility of using TC PDX models to study AFP and ß-HCG serum levels in relation to PDX growth and response to cisplatin treatment for which human protein specific ELISAs were used.
Methods
Establishing of tumor xenografts
Written informed consent was obtained before surgery from all patients of which tumor samples were used for PDX modeling. Mice were kept under pathogen free conditions and received sterilized food and water ad libitum. All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Groningen (Groningen, the Netherlands) in accordance with the approved guideline “code of practice: animal experiments in cancer research” (Netherlands Inspectorate for Health Protection, Commodities and Veterinary Public Health, 1999). Subcutaneous TC PDX models were established
as described previously7. Taking into account the heterogeneity of TC, sampling
of the tumor was assisted by a pathologist who aimed at selecting different areas guided by macroscopic examination. Histology of each tumor piece was evaluated subsequently by an experienced oncological pathologist who determined TC components. In short, primary tumors or biopsy material was cut into ~3x3x3 mm pieces and implanted on both flanks of 4-12 week old immune deficient male NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice (internal breed, Central Animal Facility, University Medical Centre Groningen). When sufficient material available, the tumor was also biobanked in liquid nitrogen using freeze media: fetal calf serum (FCS) with 5% dimethyl sulfoxyde (DMSO), paraffin embedded and snap frozen. Tumor growth was monitored by caliper measurements once a week and tumor volume was calculated using the following formula (width2
x length)/2. Once tumor volume reached approximately 1500 mm3, mice were
sacrificed and tumors harvested. These tumors (F1 generation) were used for immediate re-implantation into the next generation (F2 generation), as well as biobanked in liquid nitrogen using freeze media (FCS/5% DMSO), paraffin embedded and snap frozen.
Cisplatin treatment of PDX models
When tumors demonstrated sustained growth, mice were randomized into vehicle control or treatment groups (n=3-4 mice/group). Cisplatin (1 mg/kg or 4 mg/kg) or vehicle (saline) was administered weekly via intraperitoneal injection. Tumor growth was assessed 3 times a week using caliper measurements. All mice were sacrificed after 21 days of treatment.
Immunohistochemistry
Formalin-fixed and paraffin embedded material was cut into 4 µm sections and mounted on glass slides. Hematoxylin and eosin (H&E) stainings were used to look at tumor histology. Immunohistochemical (IHC) stainings were done for AFP
109
AFP levels do not reflect tumor response to cisplatin in testicular cancer patient-derived xenograft models (Ventana #760-2603). Tissue slides were deparaffinized in xylene and rehydrated in ethanol. Antibody incubation was done for 16 minutes at 37C. Stained sections were scanned using the NanoZoomer 2.0-HT multi slide scanner (Hamamatsu, Japan).
Evaluation of AFP and ß-HCG serum levels in PDX models
Blood samples from PDX mice were collected via retro-orbital bleeding in three
different moments: before implantation, when the tumor had reached ~200mm3
or larger and after a cisplatin treatment cycle (when applicable). After blood coagulation, samples were centrifuged for 15 minutes at 1500 rpm and serum was collected. To measure AFP and ß-HCG levels in mice serum, an ELISA kit for detection of AFP and ß-HCG in human serum of plasma was used according to manufacturer’s instructions (NovaTec Immundiagnostica, Germany). Absorbance was measured at 450 nm using an iMARK microplate absorbance reader (Bio-Rad, US). AFP and ß-HCG concentrations were determined against a standard curve provided by the ELISA kit. All samples were measured in duplicate.
Results
AFP levels are detectable in TC PDX models
We used three non-seminoma PDX models, TC1, TC4 and TC5 to study AFP and ß-HCG serum levels. TC1 was established from a tumor obtained via orchiectomy from a patient positive for AFP and ß-HCG at moment of surgery. TC4 was developed from a metastasis sample obtained from a retroperitoneal mass. This patient had low but detectable levels of ß-HCG and high AFP levels when the biopsy was taken. TC5 was established from specimen obtained via orchiectomy. This patient had elevated AFP serum levels only. Next, AFP and ß-HCG levels in mouse sera were determined when tumors had reached a
volume of approximately 200 mm3. An overview of all measurements is depicted
in Figure 1. AFP serum levels can vary largely. For instance, for PDX model TC4, AFP negative as well as positive AFP serum levels were found. This large variation is likely due to the mixed tumor phenotype of these patients. ß-HCG serum levels in mice were below detection limit in each of the models, whereas two patients had detectable ß-HCG serum levels. AFP and ß-HCG serum levels were below detection limits in mice without tumors (negative controls) excluding the possibility that the antibody could bind to any mouse serum protein.
AFP is used in the clinic to assist diagnosis of TC and follow up of patients. It is mainly expressed by the histological subtype yolk sac tumor, although it can also be produced by embryonal carcinoma components. This is in agreement with the histological analysis of our PDX models that all contained yolk sac.
AFP levels do not correlate with response to chemotherapy
To study the effect of cisplatin on serum levels of AFP, we used the chemosensitive models TC1 and TC5, and the chemoresistant model TC4. Mice were treated with either vehicle, 1mg/kg or 4mg/kg of cisplatin once a week and tumor growth was monitored for 21 days. AFP levels were detected in almost all mice bearing PDX tumors at start of treatment (Figure 2). Remarkably, AFP levels were lower or undetectable in the vehicle groups of TC1 and TC4 at day 21 compared to levels at start of treatment, although tumor volume of both models increased. In contrast, serum AFP levels were higher in TC1 after cisplatin treatment despite the decrease in tumor volume in all mice (Figure 2). In the chemoresistant PDX TC4 model, AFP levels decreased compared to start of treatment with cisplatin, while tumor volumes were increased at the end of treatment (Figure 2). In addition, AFP stainings were done on paraffin embedded material after tumors were harvested at the end of treatment (Figure 3). Vehicle and cisplatin treated TC1 and TC4 tumors were still positive for AFP and intensity of the AFP staining in both models had not changed after treatment with 4 mg/kg cisplatin. Absence of AFP in the mouse serum of TC4 after cisplatin treatment (4 mg/kg) can therefore not be explained by loss of AFP expression in the tumors.
Figure 1. AFP and HCG levels detected in human and mouse serum from PDX
models TC1, TC4 and TC5. Error bars indicate ±SEM.
Patient 1 0 1000 2000 3000 4000 5000 AFP µg/L Patient 4 Patient 5 PDX TC1 0 100 200 300 AFP µg/L PDX TC4 PDX TC5 0 3 6 9 800 1000 1200 β-HGC UI/L
Patient 1 Patient 4 Patient 5
0 1 2 3 4 5 β-HGC PDX TC1 PDX TC4 PDX TC5
< det. limit < det. limit < det. limit
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AFP levels do not reflect tumor response to cisplatin in testicular cancer patient-derived xenograft models
Start of treatment End of treatment 0 500 1000 1500 Vehicle Tumor volume (mm ³) Vehicle
Start of treatment End of treatment 0 100 200 300 400 AFP ( µg/L)
Start of treatment End of treatment 0 500 1000 1500 2000 Cisplatin 1 mg/kg Tumor volume (mm ³) Cisplatin 1 mg/kg
Start of treatment End of treatment 0 100 200 300 400 AFP ( µg/L)
Start of treatment End of treatment 0 100 200 300 Cisplatin 4 mg/kg Tumor volume (mm ³) Cisplatin 4 mg/kg
Start of treatment End of treatment 0 100 200 300 400 AFP ( µg/L) Vehicle
Start of treatment End of treatment 0 1000 2000 3000 4000 Tumor volume (mm ³) Vehicle
Start of treatment End of treatment 0 50 100 150 200 AFP ( µg/L) Cisplatin 4 mg/kg
Start of treatment End of treatment 0 200 400 600 800 Tumor volume (mm ³) Cisplatin 4 mg/kg
Start of treatment End of treatment 0 100 200 300 400 AFP ( µg/L) PD X TC1 PD X TC4
Figure 2. Comparison of biomarker levels and tumor volume. AFP levels detected
in mouse serum from PDX TC1 and TC4 at start and end of either vehicle or cisplatin treatment, and matching tumor volumes. Colored dots indicate paired samples from individual mice. Colored dots indicate paired samples from individual mice.
Figure 3. Representative images of tumors shown in (A) and (B) at 10x
magnification stained for AFP
PDX TC1 Vehicle PDX TC4 Cispl atin 4mg/kg 200 µM 200 µM 200 µM 200 µM
Discussion
Taken together, our results show AFP can be detected in the sera of mice bearing TC tumors. ß-HCG was not detectable in sera from these PDX models, even not in the PDX model established from tumor material of a strongly ß-HCG positive patient. AFP is used for follow-up of TC patients to detect relapses. Our experiments demonstrate unexpectedly that AFP levels in mice do not correlate with tumor volume. One possible explanation is the sub-optimal blood supply to the tumor. Blood circulation in subcutaneous PDX tumors might be even more aberrant than in human tumors, which are known to have abnormal blood vessel growth8. This would imply that the larger the tumors the more difficult AFP can be released into the serum. An additional explanation could be that AFP is not actively secreted by TC cells in these mice and, therefore, only when TC cells die AFP can be detected. Lastly, tumor markers can be released by the tumor as response to chemotherapy. In the vehicle treated TC1 and TC4 as well as in the cisplatin resistant TC4 mice treated with cisplatin tumor viability is high, resulting in almost no AFP release. In contrast, in cisplatin treated chemosensitive TC1 PDX that show no increase in tumor volume, an equilibrium between growth and dying cells may result in an ongoing release of AFP into the circulation. However, these results contradict with clinical observations that AFP serum reduction after chemotherapy is related to good responses in metastatic nonseminomatous
testicular cancer9. Recently, the oncogenic role of the microRNA (miR) family
miR-371 has been described in TC. A number of studies have shown that this
miR cluster can be used to diagnose TC with high sensitivity and specificity10–12.
It needs to be determined whether TC PDX models can be used to study these novel markers in the future.
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AFP levels do not reflect tumor response to cisplatin in testicular cancer patient-derived xenograft models
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