SPARC dependent collagen deposition and gemcitabine delivery in a genetically engineered mouse model of pancreas cancer

Abstract

Background: Pancreatic ductal adenocarcinoma (PDAC) is characterised by extensive matrix deposition that has been implicated in impaired drug delivery and therapeutic resistance. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that regulates collagen deposition and is highly upregulated in the activated stroma subtype with poor prognosis in PDAC patients.

Methods: Kras^G12D;p48-Cre;SPARC^-/- (KC-SPARC^-/-) and Kras^G12D;p48-Cre;SPARC^WT (KC-SPARC^WT) were generated and analysed at different stages of carcinogenesis by histological grading, immunohistochemistry for epithelial and stromal markers, survival and preclinical analysis. Pharmacokinetic and pharmacodynamic studies were conducted by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and immunohistochemistry following gemcitabine treatment (100 mg/kg) in vivo.

Findings: Global genetic ablation of SPARC in a Kras^G12D driven mouse model resulted in significantly reduced overall and mature collagen deposition around early and advanced pancreatic intraepithelial neoplasia (PanIN) lesions and in invasive PDAC (p < .001). However, detailed pathological scoring and molecular analysis showed no effects on PanIN to PDAC progression, vessel density (CD31), tumour incidence, grading or metastatic frequency. Despite comparable tumour kinetics, ablation of SPARC resulted in a significantly shortened survival in KC-SPARC^-/- mice (280 days versus 485 days, p < .03, log-rank-test). Using LC-MS/MS, we show that SPARC dependent collagen deposition does not affect intratumoural gemcitabine accumulation or immediate therapeutic response in tumour bearing KC-SPARC^WT and KC-SPARC^-/-mice.

Interpretation: Global SPARC ablation reduces the collagen-rich microenvironment in murine PDAC. Moreover, global SPARC depletion did not affect tumour growth kinetics, grading or metastatic frequency. Notably, the dense-collagen matrix did not restrict access of gemcitabine to the tumour. These findings may have direct translational implications in clinical trial design.

Keywords: Chemoresistance; Collagen; Drug delivery; Pancreatic cancer; SPARC.

Fig. 1

A: Representative H&E stainings of 10 months old SPARC^−/− mice and control wildtype mice show normal histological architecture of the pancreas. B: Pancreas weight of SPARC^−/− mice (n = 6) and control wildtype mice (n = 6)(p = .1, Mann-Whitney-U test). C: SPARC immunohistochemistry in KC-SPARC^WT mice shows robust expression in fibroblasts around PanINs (left upper panel) and cancer associated fibroblasts (right upper panel). Lower panel reveals no immunoreactivity for SPARC in KC-SPARC^−/− mice. D: H&E stainings of KC-SPARC^WT and KC-SPARC^−/−mice from early and late PanINs. E: Masson trichrome (MT) and Herovici staining in pancreata from KC-SPARC^WT and KC-SPARC^−/− reveals depletion of collagen matrix (blue), and reduction of mature collagen fibres (red) in KC-SPARC^−/− mice (7–8 months). F: Automated quantification of MT staining in KC-SPARC^WT (n = 7) and KC-SPARC^−/− (n = 10) (p < .001, Mann-Whitney-U test). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

A: Western blot analysis showing robust SPARC expression in isolated fibroblasts from KC-SPARC^WT with simultaneous collagen-I expression, whereas fibroblasts from KC-SPARC^−/− and epithelial tumour cells are devoid of SPARC expression (n = 2 for each cell line). KC-SPARC^−/− fibroblasts express collagen as SPARC is not involved in collagen synthesis but assembly and deposition. B, C: Semiquantitative analysis (score 0–3) of early (3–4 months old) and late (7–8 months old) ADM and PanIN I-II lesions in KC-SPARC^WT (n = 7/8) and KC-SPARC^−/- (n = 10/11) mice. D: Immunohistochemistry for α-SMA and Ki67 in PanIN+ADM pancreatic tissue from KC-SPARC^WT and KC-SPARC^−/−mice (3–4 months old). E: Automated quantification of α-SMA immunohistochemistry in preneoplastic tissues from KC-SPARC^WT and KC-SPARC^−/− mice (3–4 months old). F: Manual quantification of Ki67 immunohistochemistry showing no significant differences between KC-SPARC^WT and KC-SPARC^−/−mice (3–4 months old). G: Automated quantification of CD31 immunohistochemistry in preneoplastic tissues from KC-SPARC^WT (n = 7) and KC-SPARC^−/− (n = 10) mice (3–4 months old, p-values all calculated by Mann-Whitney-U test).

Fig. 3

A: PDAC frequency in KC-SPARC^WT (n = 29, 65% tumour frequency) and KC-SPARC^−/− (n = 53; 49% tumour frequency, p = .1, Fishers Exact test). B: Survival analysis of tumour bearing KC-SPARC^WT (n = 16) and KC-SPARC^−/− (n = 25) mice shows significantly reduced survival of KC-SPARC^−/−mice (280 days versus 485 days, p = .02, log-rank-test). C: Masson trichrome (MT) and picrosirius red staining in pancreatic tumours derived from KC-SPARC^WT and KC-SPARC^−/−mice shows reduced overall collagen, in particular collagen I and III (picrosirius red). D: Automated quantification of MT in tumours from KC-SPARC^WT (n = 8) and KC-SPARC^−/− (n = 17) mice (p < .05, Mann Whitney U test) E: Automated quantification of picrosirius red in tumours from KC-SPARC^WT (n = 7) and KC-SPARC^−/− (n = 18) mice. Collagen is significantly reduced in KC-SPARC^−/− mice (p = .01; Mann Whitney U test). F: Automated quantification of α-SMA immunohistochemistry in tumours from KC-SPARC^WT (n = 8) and KC-SPARC^−/− (n = 16) mice (p = .7, Mann-Whitney-U test). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

A: H&E staining from micro-metastases (circles left picture) and macro-metastases of the liver in KC-SPARC^WT mice. B: Manual quantification of macro- and micro-metastases in KC-SPARC^WT (n = 10) and KC-SPARC^−/− (n = 15). C + D Tumour tissues from KC-SPARC^WT (n ≥ 5) and KC-SPARC^−/− mice (n = 10) were assessed for gemcitabine metabolites 2 h after injection of 100 mg/kg gemcitabine by LC-MS/MS. Native gemcitabine (dFdC) and the active form of gemcitabine 2′,2′-difluorodeoxyuridine-5′-triphosphate (dFdCTP) were not significantly altered between the two genotypes. E: Manual quantification of CC3 immunohistochemistry in tumours from KC-SPARC^WT (n = 6) and KC-SPARC^−/− (n = 6) mice upon 1 dose of gemcitabine at 100 mg/kg. Mann-Whitney-U test was used for calculation of p-values.

Suppl Fig. 1

A: Western blot analysis for SPARC shows robust expression in human PSC1 and PSC2 compared to low or absent expression in human PDAC cell lines. B: Hyaluronic acid staining in KC-SPARC^WT and KC-SPARC^−/− mice. C: Automated quantification of HA staining in tumours from KC-SPARC^WT (n = 8) and KC-SPARC^−/− (n = 18) mice. D: Manual quantification of Ki67 immunohistochemistry in tumours from KC-SPARC^WT (n = 7) and KC-SPARC^−/− (n = 6) mice. E: Manual quantification of CC3 immunohistochemistry in tumours from KC-SPARC^WT (n = 7) and KC-SPARC^−/− (n = 12) mice. Mann-Whitney-U test was used for calculation of p-values.