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. 2018 Jul 24;9(57):30946-30961.
doi: 10.18632/oncotarget.25697.

Patient Derived Renal Cell Carcinoma Xenografts Exhibit Distinct Sensitivity Patterns in Response to Antiangiogenic Therapy and Constitute a Suitable Tool for Biomarker Development

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Free PMC article

Patient Derived Renal Cell Carcinoma Xenografts Exhibit Distinct Sensitivity Patterns in Response to Antiangiogenic Therapy and Constitute a Suitable Tool for Biomarker Development

Julia Schueler et al. Oncotarget. .
Free PMC article

Abstract

Systemic treatment is necessary for one third of patients with renal cell carcinoma. No valid biomarker is currently available to tailor personalized therapy. In this study we established a representative panel of patient derived xenograft (PDX) mouse models from patients with renal cell carcinomas and determined serum levels of high mobility group B1 (HMGB1) protein under treatment with sunitinib, pazopanib, sorafenib, axitinib, temsirolimus and bevacizumab. Serum HMGB1 levels were significantly higher in a subset of the PDX collection, which exhibited slower tumor growth during subsequent passages than tumors with low HMGB1 serum levels. Pre-treatment PDX serum HMGB1 levels also correlated with response to systemic treatment: PDX models with high HMGB1 levels predicted response to bevacizumab. Taken together, we provide for the first time evidence that the damage associated molecular pattern biomarker HMGB1 can predict response to systemic treatment with bevacizumab. Our data support the future evaluation of HMGB1 as a predictive biomarker for bevacizumab sensitivity in patients with renal cell carcinoma.

Keywords: HMGB1; VEGF; bevacizumab; damage associated molecular pattern; renal cell carcinoma.

Conflict of interest statement

CONFLICTS OF INTEREST JS, KK, AM, ML and ALP are salaried employees of Charles River Discovery, Freiburg, Germany. This employment does not alter our adherence to all the journal policies on sharing data and materials. All experiments were approved by the local ethics committee with written informed consent. The authors declare no competing financial interest.

Figures

Figure 1
Figure 1. Patient derived xenografts retain histological features of the original patient tumor and mimic the molecular landscape of the disease
(A) Histological features of selected PDX and corresponding patient tissue. H&E stains were prepared from FFPE samples of donor patient tissue as well as first and third passage of PDX derived thereof. All major histotypes of renal cancer are represented in the panel of 44 renal cancer PDX. The histological features like tumor/stroma ratio and differentiation remain stable when tumor tissue is implanted and passaged in immunocompromised mice. (B) 39 models were characterized by whole exome sequencing. The comparison with TCGA data revealed a significant correlation between the two data sets. Thus, our renal cancer PDX panel largely represents the molecular landscape of the human disease.
Figure 2
Figure 2. Secreted human HMGB1 levels in tumor-bearing mice do not correlate with the amount of intracellular HMGB1 or VEGF in the respective PDX but with HIF-1 alpha expression in the tumor
(A) Serum levels of human HMGB1 were determined by ELISA in tumor bearing NMRI nude mice (n = 2-5 per model, 125 in total). Serum was taken when tumor load was between 80 mm3 and 250 mm3 prior to any treatment. Mock-injected non-tumor bearing NMRI nude mice, with matching sex and age, served as negative controls (n= 6). ELISA was performed in technical duplicates. Red bars represent mean (±SEM). The grouping in high secretors and low secretors was performed by comparing the mean HMGB1 level of an individual model with the mock-injected control. If the difference was statically significant (Mann–Whitney, two-tailed) the respective model was a high secretor. A IHC was performed for human HMGB1 or HIF-1 alpha on the renal cancer PDX specific TMA including all 44 PDX models in duplicates and two renal cancer xenografts (Caki1 and MRI-H-166). Using the OSANO software the DAB+ area for each individual TMA punch was determined and plotted as blue bars (HMGB1) or grey bars (HIF-1 alpha) representing mean (±SEM) of percentage of DAB+ area of one punch. HMGB1 as well as HIF-1 alpha expression varied markedly within the panel. (B) representative images of weak, median and strong DAB signal of the HMGB1 IHC. (C) Comparison between the donor patient and its PDX model RXF 2540 showed similar expression levels of HMGB1. (D) mRNA expression of human VEGFA determined by qRT-PCR on lysates of renal cancer PDX tissue. The expression level in arbitrary units is calculated as ratio of human VEGFA vs human TBP. (E) Serum levels of HMGB1 and level of HMGB1 determined by IHC or human VEGFA as well as murine VEGFA expression determined by qRT-PCR did not correlate. The high secretors (above the read dotted line) exhibited all different levels of IHC staining intensity and qRT-PCR expression, respectively. In contrast the HMGB1 serum levels did correlate significantly (Pearson correlation coefficient 0,39, p< 0.012) with the HIF-1 alpha IHC scoring.
Figure 3
Figure 3. RCC PDX Tumors with high HMGB1 secretion show slower tumor growth
(A) Tumor volume was determined by caliper measurement biweekly in individual mice over subsequent passages for all models. Each graph depicts the median volume (±SEM) of all high (upper part) and low (lower part) HMGB1 secreting tumor models in subsequent passages 1 through 3. Passage 1 was defined as tumors growing after implantation of the patient donor tissue. (B) The passage time, defined as time between implantation and sacrifice of the animal due to tumor burden, was determined. PDX secreting high levels of HMGB1 depicted slower tumor growth over time as models with low levels of HMGB1. This phenomenon was statistically significant when comparing the passaging times of both groups (p< 0.007, Mann–Whitney test, two-tailed).
Figure 4
Figure 4. Characterization of renal cancer PDX panel by in vivo assessment of six standard of care compounds
(A) Characterization of 24 renal cancer PDX by treatment with different antiangiogenic and other targeted therapies. All investigated PDX models displayed distinct sensitivity pattern against axitinib, bevacizumab, pazopanib, sunitinib, sorafenib and temsirolimus. Two – six compounds per model were tested in monotherapy. Five mice bearing bilateral tumor implants were assigned for each treatment group including the vehicle control. Optimal T/C values are plotted and categorized into highly active (green= T/C < 32%), active (orange= T/C 32 – 70%) and resistant (red = T/C > 70%). The tumor models were grouped into secreting high levels of HMGB1 (high secretor) and low levels of HMGB1 (low secretor). (B) In parallel, once weekly, starting one day before first treatment, serum was sampled from all mice and HMGB1 level determined by ELISA. Four representative renal cancer PDX models are shown. The treatment with different targeted compounds in different renal cancer PDX models did not affect the serum level of HMGB1.
Figure 5
Figure 5. Characterization of a RCC PDX derived cell line panel under normoxic and hypoxic conditions in vitro in 2D
(A) A panel of six PDX derived and one commercially available RCC cell line were cultured under normoxic and hypoxic conditions. After 96h cells were harvested, counted and analyzed. HMGB1 protein levels were determined in the supernatant by ELISA. VEGFA RNA levels were determined by qPCR. The expression level in arbitrary units was calculated as ratio of human VEGFA vs human TBP. All cell lines showed an upregulation of HMGB1 protein in the supernatant as well as VEGFA mRNA in the tumor cells. (B) tumor cell viability was determined after treatment with Bevacizumab and Staurosporin (positive control) under normoxic and hypoxic conditions. In general, the sensitivity towards Bevacizumab treatment was higher under hypoxic conditions whereas culture conditions had no influence on the susceptibility towards Staurosporin.
Figure 6
Figure 6. The serum level of secreted HMGB1 predicts response to treatment with bevacizumab
(A) 20 established renal cancer PDX models were characterized bytreatment with anti-VEGF monoclonal antibody bevacizumab. Tumor volume was determined twice weekly until animals of the control group reached termination criteria. Group median absolute tumor volumes are plotted over time. Tumors with high secretion of HMGB1 (upper panel) responded significantly better to treatment with bevacizumab than tumors with low secretion of HMGB1 (lower panel). (B) The optimal T/C values were plotted as waterfall plot for 20 renal cancer PDX models treated with bevacizumab. The blue bars represent HMGB1 low secretor models; the green bars represent HMGB1 high secretor models. The optimal T/C values of the high secretor group were significantly lower (p < 0.0009, Mann–Whitney test, two-tailed) as the respective values of the low secretor group. (C) The optimal T/C values were plotted as waterfall plot for 20 renal cancer PDX models treated with bevacizumab. The purple bars represent human VEGFA low expressing models; the yellow bars represent human VEGFA high expressing models. The difference between the two groups was statically significant (p< 0.05, Mann–Whitney test, two-tailed). Thus, the level of secreted HMGB1 is a stronger predictive marker for bevacizumab sensitivity as the expression of human VEGFA.

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