Table of Contents  

Tuveson: Experimental approaches to ductal pancreatic cancer

Introduction

We conduct basic and applied research in pancreatic ductal adenocarcinoma (PDA) to aid the development of efficacious therapies and accurate diagnostics for this highly fatal disease. We have produced organoid and genetically engineered mouse models (GEMMs) that accurately mimic human PDA, and used them to establish fundamental molecular, cellular and pathophysiological principles about PDA, as well as to develop a preclinical therapeutics platform for the systematic evaluation of traditional and experimental agents.

In our basic studies, we evaluated the cellular and molecular evolution of PDA in our GEMM to determine that pancreatic ductal cells were positively stimulated by KrasG12D expression, and identified the induction of the Nrf2 transcription factor as important in this outcome.1 Furthermore, we found that Usp9x played an important role in KrasG12D-driven epithelial cell proliferation and transformation, providing direct targets to pursue for therapeutic strategies.2

Preclinically, we have addressed the general question of drug resistance of PDA by comparing traditional xenograft tumours to our GEMMs. Our work has led to the realization that primary PDA is an unusually hypovascular tumour, in contrast to xenografts, and we have used this observation to determine that hedgehog inhibitors increase vascularization and drug delivery in our GEMM.

The molecular hallmarks of human PDA include activating mutations in the KRAS oncogene and mutations in the tumour-suppressor genes P16/INK4A, TP53 and SMAD4.3 In addition, it has been observed that preinvasive pancreatic intraepithelial neoplasms (PanINs) initially harbour mutations in KRAS, with acquisition of tumour-suppressor gene mutations in more histologically advanced PanINs.4 My laboratory therefore established a variety of GEMMs that evolve from PanIN to invasive and metastatic PDA by expressing conditional germline alleles of KrasG12D and Trp53R172H in the mouse pancreas.5,6 We used these GEMMs to address the cellular and molecular origins of PanIN and PDA, and to evaluate the response of PDA to therapeutic challenge.

Molecular origins of preinvasive pancreatic intraepithelial neoplasm and pancreatic ductal adenocarcinoma – KrasG12D activates Nrf2

Considering that KrasG12D expression promoted the proliferation of the pancreatic ductal lineage in vivo, we investigated the pathways promoting this increased cellular fitness. Although the ectopic overexpression of oncogenic Ras in primary fibroblasts was previously shown to cause increases in intracellular reactive oxygen species (ROS) and cell cycle arrest due to oncogene induced senescence,7 we showed, in contrast, that endogenous KrasG12D expression directly lowers ROS and promotes cellular proliferation – providing a potential explanation for the differing observations between Scott Lowe’s work8 and our own. A proteomic and cell biological approach revealed that KrasG12D induced a lowering of ROS because of the increased messenger ribonucleic acid (mRNA) and protein expression of the Nrf2 (Nfe2l2) transcription factor. Nrf2 coordinates the transcription of many genes, including those primarily responsible for the detoxification of ROS by modulating the levels of active glutathione and thioredoxin.913 Nrf2 normally has a short protein half-life of < 5 minutes because of its association with the Keap1 repressor protein. Various cellular stressors that lead to the modification of the Nrf2 or Keap1 proteins can prolong the half-life of Nrf2, and somatic cancer-associated mutations in either Keap1 or Nrf2 that interfere with Keap1 binding to Nrf2, which thereby stabilize Nrf2, have been reported.1416 We found that the Nrf2 half-life was not prolonged in KrasG12D-expressing cells and also observed that mutations in Nrf2 and Keap1 were rare in human pancreatic cancer. Differences between ectopic and endogenous oncogenic Kras expression included the activation of the ROS-generating NADPH oxidase 1 by ectopic Kras. Furthermore, ectopic Ras only transiently induced Nrf2 expression, whereas this was stably sustained by endogenous KrasG12D.

Using the MEK inhibitor U0126, we found that the MAP kinase Ras effector pathway was responsible for Nrf2 mRNA induction and decreased ROS levels following expression of KrasG12D. The downstream mediator of this finding was the AP1 family member, Jun, which was increased in protein level following KrasG12D expression. Indeed, this was supported by a previous ENCODE study showing that Jun bound to the human NRF2 promoter.1

Using a constitutive Nrf2 knockout mouse, we showed that Nrf2 was also required for endogenous KrasG12D to promote proliferation in preneoplastic lung and pancreatic epithelial cells in vivo (Figure 1). Furthermore, the genetic silencing of Nrf2 in cell culture or in vivo promoted cellular senescence in the context of KrasG12D expression, suggesting that KrasG12D-expressing cells are dependent on Nrf2 during the earliest stages of cellular transformation, such as tumour initiation, and reinforcing our prior findings in fibroblasts.

FIGURE 1

Nrf2 influences disease progression in the pancreas and lung. PanIN content (top left) and proliferation (top right) are significantly decreased in Nrf2 null mice. In addition, Nrf2 null mice develop significantly fewer alveolar adenomatous hyperplasia (AAH) and bronchiolar hyperplasia (BH) (bottom left), and exhibit decreased proliferation (bottom right). Reproduced from DeNicola et al.1

HMJ-693-fig1.jpg

Interestingly, these effects could be mimicked pharmacologically by suppressing ROS metabolism with an inhibitor of glutathione synthesis, buthionine sulphoxime. In addition, the loss of Nrf2 could be compensated by treating cells with N-acetylcysteine, an antioxidant that acts as a glutathione mimetic to restore the intracellular reducing environment. This work raises the concern that antioxidants could have tumour-promoting properties in certain contexts, a concept we will pursue in future studies. Finally, our findings extended to an endogenous oncogenic BRAFV619E allele (human V600E) and the nearly physiological Rosa26 MycERT/ERT allele, suggesting a mechanism of general importance in two oncogenic pathways (Figure 2).1

FIGURE 2

Oncogenic signalling that leads to increased Nrf2 gene expression is an alternative mechanism to activate Nrf2 and thereby promote tumorigenesis. Reproduced from DeNicola et al.1

HMJ-693-fig2.jpg

Conducting transposon screens to identify new pathways that cooperate with KrasG12D in pancreatic ductal adenocarcinoma

As the progression of PanIN to PDA takes > 1 year in Pdx1-cre; LSL-KrasG12D mice, this offers the opportunity to identify additional genetic pathways that cooperate with KrasG12D to promote PDA. Indeed, such pathways could represent potential therapeutic targets. We used Sleeping Beauty (SB) transposon insertional mutagenesis screens to identify pathways that cooperate with Kras in our PanIN model. This approach utilizes two unlinked components: a SB transposase that catalyses transposon excision and mobilization, and a concatemer of transposons. Transposons mobilize as monomers and insert anywhere in the genome, although there is a preference for local jumping and insertion of transposons into AT-rich intronic areas. Owing to their design, transposons can block upstream transcription to inactivate tumour-suppressor genes or promote downstream expression to ectopically express putative proto-oncogenes (Figure 3).

FIGURE 3

The T2/onc transposon contains bidirectional gene trapping functions because of the inclusion of SV40 polyA transcriptional terminator sequences (pA) and the adenoviral strong splice acceptor sequence (SA). Also, unidirectional gene expression can be ectopically promoted by a murine stem cell virus (MSCV) 5′ long terminal repeat (LTR) sequence.

HMJ-693-fig3.jpg

Sleeping Beauty insertional mutagenesis has already been successfully applied to models of colorectal, hepatic and haematopoietic neoplasia, where implicated genes are quickly identified as a result of transposon insertion.1720 Accordingly, we generated a germline conditional SB13 transposase allele, which can be expressed upon Cre-mediated recombination in pancreatic epithelial cells. When mice harbouring the four pertinent alleles were generated (Figure 4 and Table 1), we noted that mice succumbed to pancreatic cancer at an early age. Genomic DNA was prepared by splinkerette polymerase chain reaction and high throughput sequencing used to demarcate putative insertion sites. Both the Gaussian kernel convolution framework and the Monte Carlo framework were used as statistical methods to identify over 1000 significant common insertion sites (CISs). Importantly, genes previously identified as somatically mutated in human pancreatic cancer were found to be CISs in this screen, serving as a positive control that this approach will yield useful information. For example, the transforming growth factor (TGF)-β pathway disruption was noted in > 30% of SB13 tumours (SMAD4, TGFB-R2, SMAD3, TGFB-R1), Rb1/p16Ink4a was a CIS in 20%, LKB1/STK11 in 6.5% and Fbxw7 in 24%, among others.

FIGURE 4

SB13 insertional mutagenesis promotes PanIN to PDA progression. Top left: the four alleles used in KCTSB13 mice. Both KrasG12D and SB13 expression are restricted to the pancreas. Top right: the decreased median survival of KCTSB13 mice (green, 172 days) compared with KC mice (Pdx1-cre; KrasG12D) (red, 257 days; P < 0.001). Bottom: histological evidence of PDA in KCTSB13 mice, scale bar = 100 µm. Reproduced from Pérez-Mancera et al.2

HMJ-693-fig4.jpg
TABLE 1

The frequency of mutation of human pancreatic cancer genes in the KCTSB13 tumours. Reproduced from Pérez-Mancera et al.2

Gene % tumours
SMAD3 32
SMAD4 32
TGFBR1 32
TGFBR2 32
CDKN2A 20
RB1 20
FBXW7 24
ACVR1B 19
ARID1A 19
MAP4K3 19
MII5 18.5
LKB1/STK11 6.5

Surprisingly, disruption of the X-linked deubiquitinase USP9X was found to be the major CIS that cooperates with KrasG12D to promote pancreatic cancer, with > 50% of the tumours showing disruptive insertions in this allele (Figure 5). We have begun to characterize this gene in PDA, and will use USP9X to illustrate the potential power of this approach to mine the functional genome of pancreatic cancer.

FIGURE 5

The deubiquitinase USP9X is disrupted in KCTSB13 PDA. Upper: histogram shows that the most common insertion of SB transposons occurs in the X chromosome gene USP9X, with insertions in both directions so likely inactivating. Middle: USP9X protein is absent in malignant ductal cells (arrows, lower panel) in tumours containing USP9X insertions, whereas it is present in normal ductal cells (arrows, upper panel) scale = 100 μm. Lower: USP9X mRNA is fused to the T2/Onc insertion. Reproduced from Pérez-Mancera et al.2

HMJ-693-fig5.jpg

Several functions have previously been ascribed to USP9X. First, USP9X was reported by Stefano Piccolo to regulate SMAD4 function,21 although we could not demonstrate any alteration in SMAD4 function or TGF-β responsiveness in PDA cells following USP9X loss. Additionally, Vishva Dixit published that USP9X served as a proto-oncogene in haematopoietic cancers by stabilizing MCL1.22 However, we could not demonstrate any change in Mcl1 levels following USP9X loss in PDA cells. Finally, USP9X was shown to promote the stabilization of the stress kinase ASK1 in cells;23 however, we could not reproduce this finding in PDA cells. Rather, we found that USP9X loss prevented anoikis in KrasG12D-expressing pancreatic ductal epithelial cells. An examination of cell lysates revealed that another previously described substrate of USP9X, the E3 ligase Itch,24 was affected following loss of USP9X.

Itch substrates include JunB, c-Jun, Notch1 and CXCR4, and elevation of these proteins could explain some of the effects of USP9X loss. Mechanistic studies are currently under way to address this possibility and explore other functions for USP9X. Our SB13 findings were validated with a conditional USP9X mouse that was interbred with our PanIN mice to confirm that USP9X deletion cooperated with KrasG12D to promote pancreatic cancer.

USP9X had not previously been reported as mutated in cancer, and thus we performed immunohistochemical analysis of a large number of primary human PDA samples following surgery, revealing that 10–20% of PDA tumours had extremely low or undetectable protein levels. Interestingly, patients whose tumours possessed low USP9X levels had a poor outcome following surgery. A separate cohort of patients who died as a result of PDA and underwent autopsy were analysed, and low USP9X levels correlated with a worse metastatic spread at the time of death (Figure 6). These results are consistent with a tumour-suppressor role for USP9X.

FIGURE 6

USP9X absence in PDA tumours predicts poor outcome in patients. Left: patients with high-grade PDA at the time of resection die sooner if their tumour lacks expression of the USP9X protein (blue curve, 329 days) in comparison with those positive for USP9X protein (green, 478 days; P = 0.037). Right: patients who die as a result of pancreatic cancer demonstrate either an oligometastatic pattern (fewer than 10 metastases) or a widely metastatic pattern (more than 10 metastases), and USP9X protein is frequently lost in patients with widely metastatic pancreatic cancer (54% vs. 19%; P = 0.0212). Reproduced from Pérez-Mancera et al.2

HMJ-693-fig6.jpg

Recently, we found that the USP9X protein level was lower in a series of human pancreatic cancer cell lines in comparison to normal ductal epithelial cells, and that USP9X protein levels increased substantially following treatment with the HDAC inhibitor, trichostatin A. Therefore, we hypothesize that USP9X is a previously unrecognized major tumour suppressor allele in pancreatic cancer that may be epigenetically silenced in 10–20% of early-stage cases and 50% of advanced cases to promote pancreatic cancer progression. If our hypothesis is correct, it raises the possibility that USP9X may be reactivated on treatment with modulators of the epigenome in patients whose tumour shows loss of the protein.

Developing therapeutics for pancreatic ductal adenocarcinoma

To develop effective therapies for PDA, we must consider that PDA tumours are composed of neoplastic cells and a larger number of fibroblasts, immune cells and vascular cells that comprise the stromal microenvironment. In addition, the abundant extracellular matrix also participates in a poorly understood symbiotic relationship with the neoplastic and stromal cells. We have used our GEMMs of PDA to probe these issues, and have noted that PDA contains a compromised vasculature.

Pancreatic ductal adenocarcinoma contains a deficient vasculature

Previous work in xenografts had incorrectly predicted that many agents would be active in patients with PDA, and we sought to compare such traditional xenograft models to our genetically engineered KPC model (KrasG12D; P53-R172H; Pdx-cre). We found that PDA tumours in mice and humans surprisingly contain an unusually low vascular content, with a 5- to 10-fold decreased blood vessel density when compared with xenograft models and other primary cancer types (Figure 7).25

FIGURE 7

Measurement of mean vessel density shows that KPC tumours and human tumours are poorly vascularized compared with syngeneic autografts (syn), orthotopic xenografts (ortho) and normal tissue (norm) (left). Human pancreatic tumours exhibit lower mean vascular density than the normal pancreas and chronic pancreatitis (right). Reproduced from Olive et al.25

HMJ-693-fig7.jpg

Further compounding this vascular deficiency is their location in a dense stromal matrix where many vessels are compressed. Indeed, only 30% of the PDA intratumoral vessels are well perfused by the circulating blood volume. We employed several radiological and pharmacological techniques to confirm that primary mouse PDA tumours are hypoperfused, and our findings were corroborated in patients. Although curiously adequate to support the metabolic needs of the PDA tumour, this dysfunctional vasculature nonetheless limits drug delivery and thus represents a potential barrier to therapeutic response. Furthermore, the increased perfusion of transplanted tumours when compared with our primary PDA model may explain the increased general sensitivity of xenograft tumours to various therapies (Figure 8).

FIGURE 8

Pancreatic tumours in KPC mice are poorly perfused and show reduced doxorubicin uptake detected by immunofluorescence, microbubble perfusion visualized using contrast ultrasound and Gd-DTPA extravasation measured by dynamic contrast-enhanced magnetic resonance imaging compared with transplanted tumours. Reproduced from Olive et al.25

HMJ-693-fig8.jpg

We sought methods that improve the intratumoral perfusion of pancreatic tumours and determined that the cyclopamine derivative IPI-926 caused stromal regression, vascular growth and vessel dilatation. Consequently, chemotherapeuticdelivery increased and resulted in tumour shrinkage and prolonged survival in mice. IPI-926 inhibits Smoothened in the hedgehog signalling pathway, and concurrent work by Tian et al.26 showed that this pathway was operant as a paracrine pathway in pancreatic carcinoma, with stromal fibroblasts responding to the hedgehog ligands secreted by neoplastic cells. Although encouraging, the beneficial effects of IPI-926 in mice were temporary and disease progression was associated with a return of the hypovascular state after several weeks of concurrent treatment with the chemotherapeutic agent, gemcitabine. We have subsequently pursued the cellular and molecular mechanisms controlling the stromal and vascular biology in PDA, and have initiated a clinical trial to evaluate our findings in patients. These findings were extended to stromal disruption agents including PEG-PH20, a pegylated hyaluronidase.

Stromal disruption approaches as therapeutic strategies in pancreatic ductal adenocarcinoma

As previously discussed, the lethality of PDA may, in part, be a result of poor chemotherapy delivery caused by the dense tumour desmoplastic stroma.25,27,28 Our initial preclinical therapeutic experiments determined that chemotherapy concentrations and therapeutic response can both be increased in a PDA mouse model following stromal depletion with a hedgehog pathway inhibitor.25 To further investigate the aetiology of intratumoral vascular compression and poor tissue perfusion in pancreatic cancer, and thereby find alternative agents to hedgehog pathway inhibitors, we characterized the extracellular matrix components in PDA tumours. We found that hyaluronan (HA), a major extracellular matrix component that maintains normal and neoplastic tissue structure by binding to additional proteoglycans and solvating water,29 was present at high levels in human PDA. Interestingly, compared with nine other human cancers, PDA tumours had the highest HA content (Table 2).3136

TABLE 2

Pancreatic ductal adenocarcinoma has the highest incidence of HA content compared with multiple other malignancies. Reproduced from Jacobetz et al.30

Tissue origin % cases with +HA staining
Breast Tumour (n = 117; 56%)
Normal (n = 13; 23%)
Prostate Tumour (n = 110; 46%)
Normal (n = 17; 5.8%)
Bladder Tumour (transitional cell carcinoma) (n = 106; 43%)
Normal (n = 8; 0%)
Gastric Tumour (n = 95; 42%)
Normal (n = 14; 0%)
Mesothelioma Tumour (n = 52; 37%)
Normal (n = 15; 0%)
Lung Non-small cell lung cancer (n = 169; 29%)
Small cell lung cancer (n = 21; 10%)
Normal (n = 21; 0%)
Ovary Tumour (n = 185; 12%)
Normal (n = 31; 0%)
Colon Tumour (n = 136; 28%)
Normal (n = 25; 8%)
Myeloid Multiple myeloma (n = 27; 3.7%)
Normal (n = 35; 0%)
Pancreas Ductal adenocarcinoma (n = 99; 90%)
Acini cell carcinoma (n = 2; 0%)
Mucinous adenocarcinoma (n = 5; 100%)
Papillary adenocarcinoma (n = 4; 25%)
Squamous cell carcinoma (n = 2; 100%)
Normal (n = 25; 4%)

The KPC GEMM develops PDA tumours with a dense desmoplastic matrix,6,25 and these tumours also had readily detectable levels of HA. Thus, KPC mice may be a suitable model system to determine whether or not the manipulation of HA could alter PDA tumour structure and perfusion.

Prior work showed that the tissue interstitial fluid pressure of transplanted tumours could be decreased, and the vascular patency and perfusion of such tumours improved following the digestion of HA by a recombinant polymer-modified form of PH20 hyaluronidase, PEGPH20.3740 To determine if PEGPH20 could also deplete HA from primary pancreatic tumours, PEGPH20 was administered intravenously (i.v.) to KPC mice and PDA tumours were collected over a 3-day period to assess the effectiveness of HA removal (Figure 9). HA was acutely depleted in a heterogeneous pattern following a single i.v. administration of PEGPH20, with little detectable HA remaining when evaluated up to 72 hours later.

FIGURE 9

KPC PDA tumours contain HA, and this can be rapidly depleted following intravenous administration of PEG-PH20 hyaluronidase. Scale bar = 200 μm. Reproduced from Jacobetz et al.30

HMJ-693-fig9.jpg

To evaluate the vascular patency in mice following the complete depletion of HA, KPC mice were treated twice with PEGPH20 and infused with tomato lectin prior to sacrifice (Figure 10). The treatment with PEGPH20 resulted in an increased patency of the PDA vasculature (70% vs. 30%; P = 0.028). We found that the majority of the intratumoral vessels were prominently open, and the mean vessel luminal area when measured was found to be significantly increased (p = 0.0085; see Figure 10). Therefore, similar to prior work with transplanted tumours,40 PEGPH20 treatment degrades HA and leads to significant improvements in vascular patency and mean vessel intraluminal area in PDA.

FIGURE 10

HA digestion with PEGPH20 acutely leads to vessel dilatation and increased perfusion of PDA tumours. Top left: open blood vessels are denoted by the dilated lumens that bind tomato lectin and Meca32; scale bar = 100 μm. Top right: fraction of perfused vessels is increased following PEGPH20. Bottom: The mean area of vessels increases following PEGPH20. V, vehicle treatment; P, PEGPH20 treatment. Reproduced from Jacobetz et al.30

HMJ-693-fig10.jpg

We next investigated whether or not these vascular alterations following PEGPH20 treatment translated to an improvement in the delivery of chemotherapy to PDA tissue. We used the autofluorescent properties of doxorubicin as a perfusion tracer agent and found that PDA tissue uptake was substantially increased following HA depletion (Figure 11). As gemcitabine is the chemotherapeutic agent used clinically to treat PDA patients,41 we also measured the triphosphorylated and active metabolite of gemcitabine, 2≡′,2′-difluorodeoxycytidine 5′-triphosphate (dFdCTP)42 following HA depletion. Similar to doxorubicin, dFdCTP levels were also significantly increased in PDA tissues following HA depletion with PEGPH20 (see Figure 11).

FIGURE 11

PEGPH20 treatment significantly increases the intratumoral concentrations of chemotherapeutics doxorubicin (left) and gemcitabine triphosphate (right). Reproduced from Jacobetz et al.30

HMJ-693-fig11.jpg

Furthermore, the increased levels of dFdCTP were accompanied by a decrease in intratumoral proliferation. PEGPH20 did not alter the plasma pharmacokinetic properties of gemcitabine in control mice. Therefore, HA depletion with PEGPH20 results in improved tumoral perfusion and delivery of small chemotherapeutic agents to PDA tissue.

To further characterize the perfusion of PDA tissues following HA depletion, fluorophore-conjugated dextran macromolecules were infused prior to tissue collection. Unexpectedly, both medium [molecular weight (MW) 40 kDa] and high molecular weight (MW 2 MDa) dextrans readily extravasated into PDA tissues in mice treated with PEGPH20 (Figure 12). To determine whether or not this effect was restricted to PDA tissues, multiple normal tissues that also contain HA were assessed and no increased accumulation of dextrans was noted. Therefore, both small and large molecules selectively access the interstitial space in KPC pancreatic tumours following complete HA depletion in vivo.

FIGURE 12

Large dextrans (MW 40k Da and 2 MDa) readily access the PDA tumour interstitium following treatment with PEGPH20. Scale bar = 500 μm. Reproduced from Jacobetz et al.30

HMJ-693-fig12.jpg

To determine the mechanism of increased access to PDA tissues following PEGPH20 treatment, the tumour vasculature was evaluated with scanning electron microscopy (SEM). We found that the blood vessel architecture in PDA tumours from KPC mice (Figure 13) was similar in appearance to normal pancreatic parenchymal blood vessels, with no obvious structural abnormalities characteristic of blood vessels in xenograft tumours, such as endothelial fenestrae or endothelial peri-cellular gaps.4345 Interestingly, PEGPH20 treatment did not alter the blood vessel structure in normal mouse pancreatic tissues, but did induce the significant accumulation of numerous endothelial fenestrae in KPC mouse intratumoral PDA blood vessels (see Figure 13; P < 0.0001).

FIGURE 13

PEGPH20 specifically induces fenestrae in PDA intratumoral blood vessels (SEM). This is quantified in the graph below. Reproduced from Jacobetz et al.30

HMJ-693-fig13.jpg

Therefore, PEGPH20 facilitates fenestrae formation in PDA and this correlates with the intratumoral delivery of both small molecule drugs and larger macromolecules.

We next evaluated whether or not PEGPH20 provided any therapeutic benefit to KPC mice alone and in combination with gemcitabine. Although gemcitabine monotherapy treatment caused a modest decrease in percentage tumour growth compared with vehicle (164 mm3 vs. 210 mm3; P = 0.0793), the concomitant administration of gemcitabine and PEGPH20 significantly inhibited tumour growth over 5 days of treatment when compared with gemcitabine monotherapy (104 mm3 vs. 164 mm3; P = 0.0003; Figure 14). To determine if these findings impacted upon the lethality of pancreatic cancer, mice were subsequently treated for an extended period of time with each agent as a monotherapy, with a vehicle control or with the combination of PEGPH20 and gemcitabine. Similar to the shorter time analyses, we found that monotherapeutic treatment with PEGPH20 or gemcitabine had insignificant effects on the survival of KPC mice in comparison to vehicle-treated mice (see Figure 14). Remarkably, the median survival of KPC mice treated with the combination of PEGPH20 and gemcitabine was significantly extended by 2 weeks (P = 0.0002), similar to our previous findings with hedgehog inhibition. Although gradual tumour growth occurred in most KPC mice in the PEGPH20/gemcitabine combination cohort, PEGPH20 remained effective as confirmed by the lack of HA content in PDA tissues obtained at the study end point. Therefore, PEGPH20 increases gemcitabine delivery and synergistically extends the survival of gemcitabine-treated KPC mice.

FIGURE 14

PEGPH20 synergistically works in combination with gemcitabine to suppress tumour growth (above) and prolongs survival in KPC mice (below). Reproduced from Jacobetz et al.30

HMJ-693-fig14.jpg

We recently proposed that poor chemotherapy delivery partially explained the resistant properties of pancreatic cancer, and used the hedgehog pathway inhibitor IPI926 to improve drug delivery and improve therapeutic response.25 Because the hedgehog signalling pathway has also been implicated in survival and propagation of stem-like cancer cells in PDA,26,4648 our current study with HA digestion supports the hypothesis that stromal depletion and improved drug delivery is a sufficient explanation for the results we previously reported.25 Now that two distinct mechanisms of increasing drug delivery to PDA tumours have been identified, future preclinical work should evaluate the utility of combinations of hedgehog pathway inhibitors and HA digestion.

As PEGPH20 acutely disrupts a component of the extracellular matrix in PDA tumours, this could impinge on multiple signalling pathways in the tumour to alter the proliferation and survival of both neoplastic and microenvironment cells.4952 However, when we examined the PDA tissues from KPC mice treated with PEGPH20 alone, we did not observe dramatic changes in cellular proliferation. Indeed, the primary notable findings following PEGPH20 treatment were the dilatation and increased patency of PDA vasculature, and the appearance of fenestrae in endothelial cells lining these PDA intratumoral blood vessels. The luminal expansion of the PDA vasculature by alleviating the extrinsic compression caused by HA is consistent with prior work using transplanted tumour models, in which HA promoted increased interstitial fluid pressure possibly by sequestering water and cross-linking additional proteins.40 However, the formation of intratumoral endothelial fenestrae following HA depletion and vessel dilatation was unanticipated. The precise aetiology of the endothelial fenestrae will be investigated further, and may include the increased sensitivity of the re-expanded PDA intratumoral blood vessels to circulating factors, the release of additional sequestered vascular permeability factors from the ECM or a biophysical response to the rapid change in tumour matrix architecture. Nonetheless, the increased vascular patency and volume coupled with the endothelial fenestrae should facilitate the delivery of gemcitabine and other drugs to the tumour stroma in PEGPH20-treated KPC mice. Furthermore, the finding that high-MW species can now access the tumour stroma represents an additional opportunity to evaluate whether or not PEGPH20 can alter the response of PDA tumours to agents such as targeted antibodies and albumin–chemotherapy conjugates. Therefore, HA restricts the functional properties of the intratumoral vasculature and represents a novel stromal therapeutic target to consider for patients with pancreatic cancer. As is true of most therapeutics that we evaluate in our model, PEGPH20 is formulated for clinical investigation, and early results from a phase 1 trial have identified a recommended phase 2 dose.53 Investigational trials that combine PEGPH20 with gemcitabine are a logical extension of this work for patients with PDA and will be pursued.

Developing future studies in PDA using organoids

The KRAS oncogene is mutated in > 90% of pancreatic cancers, making it an attractive therapeutic target.54 Unfortunately, attempts to generate potent Kras inhibitors have failed. Because there are no effective agents that directly target the Kras protein,54 we recently developed pancreatic organoid models in efforts to provide high-throughput screening methods for drugs that target primary PDA cells.55 The organoids and GEMMs will enable future studies to identify new biological dependencies for this deadly disease, ultimately improving the care of patients.

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