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. 2018 Apr 10;9(27):19294-19306.
doi: 10.18632/oncotarget.25022.

Patient derived xenografts (PDX) predict an effective heparanase-based therapy for lung cancer

Amit Katz  1 , Uri Barash  2 , Ilanit Boyango  2 , Sari Feld  2 , Yaniv Zohar  3 , Edward Hammond  4 , Neta Ilan  2 , Ran Kremer  1 , Israel Vlodavsky  2
Affiliations

Patient derived xenografts (PDX) predict an effective heparanase-based therapy for lung cancer

Amit Katz et al. Oncotarget. .

Abstract

Heparanase, the sole heparan sulfate (HS) degrading endoglycosidase, regulates multiple biological activities that enhance tumor growth, metastasis, angiogenesis, and inflammation. Heparanase accomplishes this by degrading HS and thereby facilitating cell invasion and regulating the bioavailability of heparin-binding proteins. HS mimicking compounds that inhibit heparanase enzymatic activity were examined in numerous preclinical cancer models. While these studies utilized established tumor cell lines, the current study utilized, for the first time, patient-derived xenografts (PDX) which better resemble the behavior and drug responsiveness of a given cancer patient. We have previously shown that heparanase levels are substantially elevated in lung cancer, correlating with reduced patients survival. Applying patient-derived lung cancer xenografts and a potent inhibitor of heparanase enzymatic activity (PG545) we investigated the significance of heparanase in the pathogenesis of lung cancer. PG545 was highly effective in lung cancer PDX, inhibiting tumor growth in >85% of the cases. Importantly, we show that PG545 was highly effective in PDX that did not respond to conventional chemotherapy (cisplatin) and vice versa. Moreover, we show that spontaneous metastasis to lymph nodes is markedly inhibited by PG545 but not by cisplatin. These results reflect the variability among patients and strongly imply that PG545 can be applied for lung cancer therapy in a personalized manner where conventional chemotherapy fails, thus highlighting the potential benefits of developing anti-heparanase treatment modalities for oncology.

Keywords: PDX; PG545; heparanase; lung cancer; metastasis.

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Conflict of interest statement

CONFLICTS OF INTEREST Edward Hammond is employed by Zucero Therapeutics, Darra, Queensland, Australia. All other authors have no potential conflicts of interest to declare.

Figures

Figure 1
Figure 1. Overexpression of the heparanase gene stimulates lung carcinoma tumorigenesis
HCC-827 lung carcinoma cells were stably infected with the heparanase gene (Hepa) and examined for heparanase activity vs mock (Vo) transfected cells (A). Cell lysates were incubated with sulfate labeled ECM. Labeled HS degradation fragments released into the incubation medium were subjected to gel filtration on Sepharose 6B column. Inset: immunoblotting for heparanase in Hepa- vs. Vo cells. (B, C) Cell invasion. Heparanase (Hepa) and mock (Vo) transfected HCC-827 cells (1 × 105) were plated onto Matrigel-coated 8-μm transwell filters. Invading cells adhering to the lower side of the membrane were visualized (B) and counted (C) after 16 h. (D) Colony formation. HCC-827 cells were seeded (2 × 103/35 mm dish) in soft agar and grown for 2 weeks. The number of colonies produced by Hepa vs. control Vo cells was quantified and is shown graphically. (E, F) Tumor growth. Heparanase (Hepa) and mock (Vo) transfected HCC-827 cells (2 × 106) were injected subcutaneously to NOD/SCID mice. Tumor volume was calculated from external caliper measurements (E, upper panel). At the end of the experiment on day 50, tumors were resected and weighed (E, lower panel). (F) Effect of PG545. HCC-827 cells (2 × 106) were injected subcutaneously to NOD/SCID mice. A group of mice (n = 7) was treated with PG545 (20 mg/kg; once a week) and the control group (Con) with vehicle alone. Tumor volume (upper panel) and weight (lower panel) were measured as described above. G. Immunoblotting. HCC-827 (left) and A-549 (right) cells (2 × 106) were untreated (Con) or treated with PG545 (PG; 50 mg/ml, 24 h) and subjected to immunoblotting applying anti-phospho Akt (pAkt, upper panels), anti-Akt (second panels), anti-p27 (third panels) and anti-actin (lower panels) antibodies. Note reduced Akt phosphorylation (bar graphs representing densitometric analysis of pAkt relative to total Akt) and up-regulation of p27 in response to PG545.
Figure 2
Figure 2. Patient derived xenografts (PDX) maintain characteristics of the respective primary tumors
(A, B ) Heparanase activity. Portions of the parent (primary) lung tumor biopsy specimen (# 1709) and the respective PDX (A), and of the primary PDX and resulting metastasis (B) were extracted, homogenized, and examined for heparanase enzymatic activity as described in ‘Methods’. The other portion was fixed subjected to immunostaining for heparanase (Hepa; C). Note heparanase immuno-reactivity in the lung tumor (C, lower panel) but not in adjacent normal lung tissue (C, upper panel). Original magnifications: ×100. (D) Lymph-node metastasis of PDX 1709 was stained with hematoxylin & eosin (upper panel), anti-heparanase antibody (Hepa, middle panel), and anti-HLA (lower panel) to clearly label human cells within the mouse lymph node. Original magnifications: ×100. (E) Sections of the primary 0906 tumor and the resulting PDX were subjected to immunostaining applying anti-heparanase (left), anti-smooth muscle actin (SMA; middle) and anti-cytokeratin 8 (CK8; right) antibodies. A similar expression pattern of heparanase, SMA and CK8 was noted in the parent primary tumors and the respective PDX. Magnification: left and right panels ×100; middle panels ×25.
Figure 3
Figure 3. PG545 attenuates the growth of patient derived lung cancer xenografts to various degrees
Fresh tumor biopsies were collected from surgery, cut into small pieces and implanted (s.c) into the flank of NOD/SCID mice. Once tumors reached 1–1.5 cm in diameter, tumors were excised, cut into small fragments and re-implanted into NOD/SCID mice. When the tumors became palpable, mice were divided into control (Con) untreated group and study group (PG) that was treated with PG545 (20 mg/kg, once a week). Tumor volume was calculated from external caliper measurements (upper panels). At the end of the experiment, tumors were resected, photographed (insets) along with macroscopic lymph node metastases (Con Mets, PG Mets), and weighed (lower panels). Tumor growth inhibition was categorized as ‘good’ response (A, 1709), intermediate response (B; 2210), or poor response (C, 1810), representing more than 80%, 50–79%, or less than 50% inhibition, respectively. Notably, when spontaneous metastasis to lymph nodes was observed, PG545 not only inhibited tumor growth but also practically prevented tumor metastasis (A, B, insets). X represents no detectable metastases in the indicated treatment regimen.
Figure 4
Figure 4. PG545 and cisplatin differentially attenuate the growth of patient derived lung cancer xenografts
Fresh patient derived tumor biopsies were cut and implanted (s.c) into the flank of NOD/SCID mice. Once tumors reached 1–1.5 cm in diameter, tumors were excised, cut and re-implanted into NOD/SCID mice. When the tumors became palpable mice were divided into control (Con) untreated group and study groups that were treated with PG545 (PG, 20 mg/kg, once a week), cisplatin (Cis, 3 mg/kg, every two weeks) or both. Tumor volume was calculated from external caliper measurements (upper panels). At the end of the experiment, tumors were resected, photographed along with macroscopic metastases (Con-Mets, Cis-Mets, PG-Mets), and weighed (lower panels). PDX #1709 responded well to PG545 in term of inhibiting tumor growth and tumor metastasis (X represents no detectable metastases in the indicated treatment regimen), but did not respond to cisplatin (A), whereas PDX 0906 responded well to cisplatin but only partially to PG545 (B). *= p values vs Control; **= p value vs PG545, for both Cis and PG+Cis.
Figure 5
Figure 5. PG545 as neoadjuvant
(A) Schematic diagram of the experiment. PDX was established from a spontaneous metastasis of PDX 1709 to lymph node (1709-Met). The 1709-Met PDX was implanted in 18 NOD/SCID (D1). Once palpable (day 7), mice were left untreated as control (n = 12) or were treated with PG545 (n = 6; 20 mg/kg once a week). Three weeks later, when tumors reached a diameter of 10–15mm, tumors were resected and weighed (B). Six (out of 12) of the former control mice received PG545 (PG post-Op) while the other 6 mice were kept untreated (Con). One month later, mice were sacrificed and examined for local recurrence and lymph node (LN) metastases (C). X represents no detectable tumors or tumor metastases in the indicated treatment regimen. An example of lymph node metastasis in this model is shown in (C, inset). Note that unlike the original 1709 PDX, the 1709-Met PDX does not respond well to PG545 (B). However, PG545 is highly effective in prevention of tumor recurrence and metastasis if present before tumor resection (neoadjuvant).
Figure 6
Figure 6. PG545 affects tumor and stromal cells
PDX 1709 tumors were treated with vehicle (Con) or PG545 (PG, 20 mg/kg, once a week). At the end of the experiment, tumors were resected and subjected to histological examination and immunostaining with antibodies directed to CD31 (a marker for vascular endothelial cells, upper panels), phospho Erk (pErk; second panels) and against F4/80 (a marker of macrophages; lower panels). Magnification: ×100, except the third panels (F4/80): ×25.
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