Superior therapeutic efficacy of nab-paclitaxel over cremophor-based paclitaxel in locally advanced and metastatic models of human pancreatic cancer

Abstract

Background: Albumin-bound paclitaxel (nab-paclitaxel, nab-PTX) plus gemcitabine (GEM) combination has demonstrated efficient antitumour activity and statistically significant overall survival of patients with metastatic pancreatic ductal adenocarcinoma (PDAC) compared with GEM monotherapy. This regimen is currently approved as a standard of care treatment option for patients with metastatic PDAC. It is unclear whether cremophor-based PTX combined with GEM provide a similar level of therapeutic efficacy in PDAC.

Methods: We comprehensively explored the antitumour efficacy, effect on metastatic dissemination, tumour stroma and survival advantage following GEM, PTX and nab-PTX as monotherapy or in combination with GEM in a locally advanced, and a highly metastatic orthotopic model of human PDAC.

Results: Nab-PTX treatment resulted in significantly higher paclitaxel tumour plasma ratio (1.98-fold), robust stromal depletion, antitumour efficacy (3.79-fold) and survival benefit compared with PTX treatment. PTX plus GEM treatment showed no survival gain over GEM monotherapy. However, nab-PTX in combination with GEM decreased primary tumour burden, metastatic dissemination and significantly increased median survival of animals compared with either agents alone. These therapeutic effects were accompanied by depletion of dense fibrotic tumour stroma and decreased proliferation of carcinoma cells. Notably, nab-PTX monotherapy was equivalent to nab-PTX plus GEM in providing survival advantage to mice in a highly aggressive metastatic PDAC model, indicating that nab-PTX could potentially stop the progression of late-stage pancreatic cancer.

Conclusions: Our data confirmed that therapeutic efficacy of PTX and nab-PTX vary widely, and the contention that these agents elicit similar antitumour response was not supported. The addition of PTX to GEM showed no survival advantage, concluding that a clinical combination of PTX and GEM may unlikely to provide significant survival advantage over GEM monotherapy and may not be a viable alternative to the current standard-of-care nab-PTX plus GEM regimen for the treatment of PDAC patients.

Figure 1

Characteristics of orthotopically implanted PDAC tumours in mice and therapeutic efficacy of treatment regimens. Upon implantation in mouse pancreas, untreated Panc265 tumour showed more aggressive tumour growth and metastatic dissemination in mouse organs. (A) Photographs of resected liver, spleen and mesenteric lymph nodes from an untreated Panc265 tumour harbouring mouse showing prominent metastatic lesions in organs (upper panel). Yellow arrows point towards metastatic lesions. Representative photomicrographs (H&E, × 40) showing primary tumour growth in mouse pancreas, metastatic dissemination in liver, spleen and lungs. Black arrows point towards metastatic deposits in organs. (B) Gross morphology representative of the resected primary tumours at the termination of efficacy experiment (D28). Primary tumour volumes in different treatment regimens are shown in (C). Data represent mean primary tumour volumes ±s.e.m. N=9–10 mice per group in Panc185 and 8–10 mice per group in Panc265. Baseline mean tumour volumes (N=5) are shown on the left side of graphs. Statistical comparison of primary tumour volumes and P-values are shown in Supplementary Table S3. (D) Representative photomicrographs (H&E, × 20) of primary tumour sections. Histological evaluation of Panc185 and Panc265 tumours showed moderately differentiated and poorly differentiated pancreatic ductal adenocarcinoma, respectively. Blue arrows point towards desmoplastic stroma. Red arrows point towards areas of neutrophil infiltrate. Green arrows point towards normal pancreatic acinar cells.

Figure 2

Treatment effect of agents alone and in combinations on tumour cell proliferation and tumour vascularity. The nab-PTX plus GEM combination was highly effective in controlling tumour cell proliferation, as evidenced by reduced number of Ki-67-stained carcinoma cells. Tumours harvested from four to five mice of Panc185 and Panc265 on day 28 (first experiment) were processed and stained with Ki-67 or CD31 antibodies. Ki-67, a nuclear protein, which is expressed in the proliferating carcinoma cells is visualised as dark brown. There was no difference in various sized blood vessel density in tumours harvested from drug-treated animals compared with tumours in the untreated animals. CD31-stained endothelial cells in blood vessels are visualised as brown stain. (A, B) Representative photomicrographs of Ki-67 or CD31 stained tumour sections (× 20).

Figure 3

Treatment effect of agents alone and in combinations on desmoplastic tumour stroma. Nab-PTX monotherapy or combined with GEM robustly depleted the dense tumour stroma as evidenced by less number of collagen fibres in the primary tumours. We performed immunohistochemistry on primary tumours harvested from untreated or treated with GEM, PTX, nab-PTX and combinations of GEM plus PTX or GEM plus nab-PTX (D28 samples, first experiment). The fibrotic tissue is visualised as blue (Mason's trichrome) or brown stain (Collagen IV). (A, B) Representative photomicrographs of Mason's trichrome and Collagen IV stained tumour sections (× 20).

Figure 4

Treatment effect of regimens on the survival of mice with established PDAC. Nab-PTX monotherapy and in combination with GEM significantly improved the survival of mice with orthotopically implanted human PDAC. (A and B) Kaplan–Meir survival curves of mice with orthotopically implanted Panc185 tumour and Panc265 tumours. Briefly, tumour harbouring mice were treated with GEM, PTX, nab-PTX and combinations of GEM and PTX or GEM and nab-PTX as described in the study design. Survival data were analysed using GraphPad Prism 6 software. P-values for survival differences were calculated using the log-rank (Mantel-Cox) test. Tables shown on the right side of figure panels depict the median survival days and statistical significance. N=10 mice per group in both Panc185 and Panc265 PDXs.

Figure 5

Acute effect of taxanes on plasma and intratumour paclitaxel concentrations and desmoplastic stroma. Nab-PTX treatment resulted in higher intratumour paclitaxel concentration, higher paclitaxel tumour plasma ratio and facilitated depletion of tumour desmoplastic stroma, compared with PTX treatment. For the quantitation of plasma and intratumour paclitaxel, mice with established Panc265 orthotopic tumours were either untreated or treated with PTX/nab-PTX once daily, i.v. for five consecutive days as described in the Materials and Methods. Blood was collected and tumours were resected 2h post the last dose of PTX or nab-PTX on day 5 (N=9 mice/group). Tumour and plasma paclitaxel concentrations were analysed by LC/MS/MS. (A) Scatter plots showing plasma paclitaxel concentrations of animals administered with PTX or nab-PTX. Plasma paclitaxel concentrations of PTX was not statistically significant compared with nab-PTX (P=0.8357). As shown in panel B, nab-PTX treatment was associated with significantly higher (30.158%) tumour paclitaxel concentrations compared with PTX treatment (**P=0.0014). The paclitaxel tumour plasma ratio was significantly higher (1.98-fold) in animals administered with nab-PTX compared with PTX treatment (C, *P=0.0311). In order to determine the acute effect of taxanes on tumour stroma, mice with established Panc185 and Panc265 orthotopic tumours were either untreated or treated with PTX/nab-PTX once daily, i.v. for five consecutive days as described in the Materials and Methods. Tumours were resected 24 h post the last dose of PTX or nab-PTX on day 5 (N=5 mice/group). (D) Representative photomicrographs (× 20) of Masson's trichrome and Collagen IV staining performed on formalin-fixed paraffin-embedded primary tumour tissues. Treatment with nab-PTX resulted in a higher depletion of tumour desmoplastic stroma compared with PTX treatment. Fibrotic tissue is visualised as blue, nuclei are stained dark red/purple and cytoplasm is stained red/pink (Masson's trichrome). Collagen IV enriched fibrotic areas are visualised as brown stain.