Tumor Priming by SMO Inhibition Enhances Antibody Delivery and Efficacy in a Pancreatic Ductal Adenocarcinoma Model Free

Pancreatic cancer is the fourth most common cause of cancer mortality in the United States (1). Pancreatic ductal adenocarcinoma (PDAC) is the most prevalent and has a dismal 5-year survival of 6%. Chemotherapy provides only marginal therapeutic benefit (1, 2), and several factors conspire to limit its efficacy. Extensive expansion of extracellular matrix (ECM) and stromal cells creates a barrier to drug delivery (3–5), and inadequate drug exposure of tumor cells is a key contributor to therapeutic failure. In addition, PDAC tumors are hypovascular and hypoperfused, which, along with high interstitial fluid pressure (IFP) and tumor solid stress, limits drug delivery (5–10).

Drug delivery barriers in PDAC hinder tumor access of macromolecules such as mAbs to a greater extent than small-molecule drugs. Following convective delivery to the tumor via the blood, mAb must extravasate and then distribute throughout the tumor. Diffusion rates of macromolecules in tissues are far lower than those of small-molecule drugs, and the extensive ECM produced by stromal cells constitutes a physical barrier to intratumor distribution (5, 7, 11). These factors together hinder establishment of effective tumor concentrations of macromolecular drugs. Notably, delivery of inadequate drug concentrations may exacerbate treatment resistance by selecting for therapy-resistant cells (12, 13).

Strategies that target signaling pathways supporting stromal elaboration represent a potential approach to compromise the drug delivery barriers in PDAC. Sonic hedgehog (sHH) signaling promotes proliferation of tumor stromal cells and stimulates synthesis of ECM, which hinders drug penetration (4, 14–17). Effects of sHH signaling upon tumor microvessel density (MVD) and angiogenesis are complex. Reports indicate that sHH signaling promotes angiogenesis (18–21), and that Smoothened (SMO) inhibitors of sHH signaling (sHHI) mediate transient elevation of tumor MVD, perfusion, permeability, as well as delivery of chemotherapeutic agents (4) and nanoparticulate drug carriers (22). The effects of sHH signaling inhibition appear to be dose-dependent, with partial inhibition increasing both tumor growth and the angiogenic influence of tumor-associated fibroblasts, whereas complete inhibition reduces tumorigenesis and angiogenesis (23). Contrasting observations suggest that stroma restrains tumor growth (24–26) and led us to hypothesize that optimal selection of the dose and duration of sHHI treatment may be essential for successful deployment of sHHI to target tumor drug delivery barriers.

Our objective was to test the hypothesis that a temporal “tumor priming” window, established by sHHI pretreatment, could compromise the barrier to therapeutic mAb deposition by increasing tumor perfusion and enhancing intratumor distribution. Numerous sHHIs have been developed, and two are clinically approved. NVP-LDE225 (Sonidegib, Novartis) was chosen for these studies (27). Because most cell line–based pancreatic cancer models lack the desmoplasia typical of PDAC, patient-derived xenograft (PDX) PDAC models were selected that recapitulate the desmoplasia and low vascularity of human PDAC (28). Cetuximab was chosen as the proof-of-concept tumor-targeting mAb because approximately 85% of PDAC tumors overexpress the EGFR (29). Erlotinib, a small-molecule EGFR tyrosine kinase inhibitor, is approved for PDAC treatment and validates the concept of EGFR signaling as a therapeutic target (30, 31). However, cetuximab has not shown efficacy in phase III PDAC trials (32). Our approach was to assess not only the magnitude by which tumor priming can enhance mAb delivery and intratumor distribution, but also whether priming increases mAb antitumor efficacy.

PDX PDAC tumors were established at Roswell Park Comprehensive Cancer Center (28). Fragments (8 mm³) from donor mice (passages 4–9) were implanted subcutaneously in the abdominal wall of anesthetized 18 to 20 gm CB17 SCID mice. When tumors reached 150 to 500 mm³, mice were randomized into groups having statistically indistinguishable starting volumes (Kruskal–Wallis test with Dunn multiple comparisons test), and treatments were initiated. Tumor volume was calculated as:

All procedures were approved by the Roswell Park Institutional Animal Care and Use Committee. Tumor DNA sequencing for driver mutation analysis was performed by Omniseq, Inc., and the data have been deposited in the Sequence Read Archive of the National Center for Biotechnology Information, U.S. National Library of Medicine as BioProject Accession #PRJNA554899, https://www.ncbi.nlm.nih.gov/bioproject/PRJNA554899.

NVP-LDE225 (Novartis), obtained as a gift or purchased as the diphosphate salt (ChemieTek), was formulated in 0.5% methylcellulose/0.5% Tween80 (ThermoFisher; ref. 33) and administered daily by oral gavage. Cetuximab (anti-EGFR) and nontargeted mAb rituximab (anti-CD20) were obtained from a pharmacy. Dr. J. Balthasar (UB) provided nontargeted mAb 8C2 (anti-topotecan).

Antibodies were labeled with N-hydroxysuccinimidyl esters of Alexa Fluor 350 or 647 (ThermoFisher) according to the manufacturer's instructions. Unconjugated dye was removed by dialysis and Centriprep Filter Units (30K NMWL, EMD Millipore). The final fluorophore:mAb ratio was approximately 6:1.

EGFR and phospho-EGFR (pEGFR) expression was detected by IHC in paraffin-embedded tumor sections fixed in fresh 4% paraformaldehyde. Primary antibodies were anti-EGFR (ab2430, Abcam) and anti-pEGFR (#2235, Cell Signaling Technology). After deparaffinization and rehydration, antigen was retrieved in Tris - EDTA (10 mmol/L - 1 mmol/L) pH 9 buffer at 95–100°C for 20 to 30 minutes. Slides were blocked (1 hour, 18°C) with TBS containing 10% normal goat serum (NGS) and 1% BSA. Primary antibody (1:400 anti-EGFR; 1:100 anti-pEGFR) in blocking buffer was incubated overnight at 4°C. The secondary antibody was the Vectastain elite ABC system for rabbit IgG (Vector Laboratories), used per the manufacturer's protocols.

CD31, Ki67, and collagen I were quantified in frozen sections by immunofluorescence. Fixatives were zinc formalin (Sigma-Aldrich) for CD31, and cold acetic acid/ethanol for Ki67 and collagen I. After blocking with Dulbecco's PBS containing 10% NGS for 1 hour, primary antibody dilutions (1:30 anti-CD31 #550274, BD Biosciences; 1:200 anti-collagen I ab34710, Abcam; 1:300 anti-Ki67 ab15580, Abcam) were incubated at 4°C overnight. Secondary antibodies were DyLight-649–labeled anti-rat IgG for CD31 (#072-05-16-06, KPL Inc.) and Alexa Fluor 488–conjugated anti-rabbit IgG H&L (ab150073, Abcam) for collagen I and Ki67. Slides were mounted with proLong gold anti-fade reagent with DAPI (ThermoFisher).

Frozen sections for evaluation of hyaluronan (HA) content were fixed with acetic acid/ethanol and probed with biotinylated hyaluronic acid binding protein (#385911, EMD Millipore) and DyLight-488 streptavidin (Vector; ref. 34).

Functional vessel density was quantified by i.v. injection of 100 μg FITC-labeled Lycopersicon esculentum lectin (FL-1171, Vector) at 2 mg/mL 10 minutes before euthanasia. Tumor cell:microvessel distance was measured as the average distance from each microvessel (CD31⁺ endothelial cells) to the five nearest tumor cells (4) for all microvessels in 5 microscopic tumor fields per animal.

Tumor RNA was isolated per the manufacturer's instructions (RNeasy Minikit; Qiagen) using RNase inhibitor N808-0119 (Applied Biosystems) and kit 4368814 (Applied Biosystems) for RT. Real-time PCR utilized TaqMan primer probe sets in Master Mix buffer (Applied Biosystems) and an Mx3005p qPCR system (Stratagene). The primer sets were Hs01110766_m1 and Mm00494645_m1 for human and mouse Gli1, and Hs00375863_m1 and Mm00496299_m1 for human and mouse TATA box binding protein-associated factor (Applied Biosystems). Expression levels were normalized by total RNA.

A Zeiss AxiophotZ1 fluorescence microscope was used for quantitative imaging, using a 20x objective to acquire images. Analysis employed ImageJ v.1.47v (http://rsbweb.nih.gov/ij/). Five to seven fields were selected randomly from each section using an automated stage. A differential interference contrast (DIC) image was taken with each fluorescence field to permit image segmentation. Cetuximab and Ki67 data were normalized by the number of DAPI⁺ nuclei per field. HA and collagen I were normalized by the stromal area/field.

Tumor IFP was measured as described (35) using a pressure transducer (SPR524; Millar Instruments) hydraulically coupled to a 23½-gauge needle catheter and calibrated using a manometer over the range of 0 to 30 cm water. The needle was inserted into the tumor under anesthesia, and the pressure was allowed to equilibrate. Because IFP increases from the tumor periphery to the core, measurements were made at increasing depths, and the highest measured pressure was recorded.

Desmoplasia is a prominent characteristic of PDAC that hinders drug penetration and intratumor distribution. Most PDAC models of cell line origin lack desmoplastic stroma, which potentially contributes to discrepancies between preclinical and clinical outcomes (4, 36). Low-passage PDAC PDX tumors were evaluated for their retention of desmoplastic stroma and recapitulation of features of the original patient tumor through passage in immunocompromised mice (ref. 28; Supplementary Fig. S1). Supplementary Table S1 reports mutation profiles for these models. The median MVD for representative PDAC PDX tumors having moderate to dense stroma was 10.4 (tumor #18254), 26.6 (#12459), 40.0 (#14312), and 63.7 (#18269) microvessels/mm². The microvessel-to-tumor cell distance is low in cell line PDAC models but high in clinical PDAC (4). The average distance of the most vascularized PDAC PDX (#18269) was 20 to 60 μm (Fig. 1A), similar to clinical PDAC tumors (4).

Treatment of PDAC tumor–bearing mice with SMO antagonists suppresses Gli1 expression, compromises stromal integrity, and increases drug delivery (4, 15, 18, 22). Gli1 was therefore selected as a sHH signaling biomarker (27, 37) and evaluated in numerous PDAC PDX tumors in sHHI-treated animals using species-specific TaqMan qRT-PCR probes to differentiate stromal Gli1 expression (mouse) versus adenocarcinoma GLI1 (human) expression. Little human GLI1 was detected in these PDAC PDX tumors, and its abundance changed slightly with SMO antagonist dosing (Supplementary Fig. S2). Previous reports suggest that GLI1 expression is regulated independently of sHH signaling in most PDAC tumors having KRAS gene mutations (38, 39). In contrast, murine Gli1 expression was abundant, and all PDX tumors responded to 40 and 80 mg/kg/day NVP-LDE225 with time-dependent Gli1 suppression (Fig. 1B; Supplementary Figs. S2B and S3). Within 3 days of initiating daily oral dosing at 40 mg/kg/day, a dose that did not inhibit tumor growth significantly with short treatment courses (22), tumor Gli1 expression was reduced to 5% of control at the time of plasma drug trough concentrations (24 hours; Fig. 1B), and inhibition was sustained through 7 days of dosing. On the 10th day of dosing, Gli1 expression at the plasma trough time had recovered slightly, suggesting onset of pharmacodynamic tolerance to continuous dosing, but the trend was not significant. Forty-eight hours after the last sHHI dose, Gli1 expression recovered to pretreatment levels.

The time-dependent enhancement of tumor perfusion/permeability mediated by sHHI was investigated in several of the PDX PDAC models using 80 nm fluorescent nanoparticles as permeability probes (22). To varying degrees, most tumors showed time-dependent changes in nanoparticle deposition after 4 to 7 days of daily oral sHHI dosing (Supplementary Fig. S4).

PDX #18269, which has extensive desmoplasia (Supplementary Fig. S1) and low vascular permeability (22), was selected for more detailed investigation of tumor responses to sHHI treatment. Effects upon cell proliferation (Ki67 expression) in tumor and stromal regions were investigated by immunostaining over 20 days of sHHI dosing (Fig. 1E). Tumor cell regions contained the greatest abundance of proliferating cells; in control animals, 52% of cells were Ki67⁺ (Fig. 1C), with some regions 100% positive. Stromal regions were low in proliferation, averaging 13% Ki67⁺ cells (Fig. 1D). For both tumor and stromal regions, treatment with sHHI reduced the number of proliferating cells. In stromal regions, the fraction of Ki67⁺ cells decreased significantly after 3 days of treatment and remained suppressed through 20 days of sHHI dosing (Fig. 1D). In tumor cell regions, proliferation was statistically unchanged after 3 days of treatment, but declined after 7 to 8 days, and regions having high fractions of proliferating tumor cells became less abundant (Fig. 1C). The delayed response suggested an indirect effect of sHHI treatment upon tumor cell proliferation.

The hypovascularity and poor perfusion of PDAC tumors limits delivery of small-molecule drugs, and previous reports indicated that sHH signaling inhibition increased PDAC MVD and permeability in a time- and dose-dependent manner (4). During 10 days of dosing with 40 mg/kg/day NVP-LDE225, CD31 staining showed no significant change in MVD in PDX #18269 (Fig. 2A; Supplementary Fig. S5A and S5B). Total MVD does not necessarily reflect tumor perfusion, because of vascular tortuosity, loss of patency, and malformations (40, 41). Therefore, functional microvessels were evaluated by i.v. injection of fluorescent Lycopersicon esculentum lectin 10 minutes before sacrifice. After 3 days of sHHI treatment at 40 mg/kg/day, functional MVD was unchanged relative to controls (Fig. 2B), but more than doubled after 7 days of treatment (Fig. 2B). In addition, the fraction of microvessels that were functional also was elevated on day 7 of treatment (Fig. 2C). After 10 days of treatment, when qRT-PCR analysis showed a slight recovery of Gli1 expression (Fig. 1B), the number of functional microvessels had declined to levels statistically indistinguishable from controls. However, the fraction of functional microvessels remained elevated on day 10 and on the 4 days after withdrawal of the sHHI (Fig. 2C; P ≤ 0.001), suggesting a delay in restoration of tumor barrier properties after cessation of treatment.

The dose dependence of sHHI effects on functional vessel abundance also was investigated. With 80 mg/kg/day NVP-LDE225, functional vessel density increased transiently and earlier than with 40 mg/kg/day, peaking and then declining after 3 days of dosing (Supplementary Fig. S5C and S5D). Overall, the elevation of functional vessel density with higher sHHI doses was shorter in duration.

Desmoplasia not only creates a physical drug delivery barrier, but also contributes to elevated tumor IFP and tissue solid stress (5–7). Intratumor pressures can collapse vessels and cause outward convective flow that could hinder antibody deposition (6, 8–10). Daily sHHI treatment with 40 mg/kg/day resulted in a significant (P ≤ 0.05) decline in peak IFP at day 3, which continued to decline over 11 days of dosing, when it reached a nadir (Fig. 3A). Temporally, the decline in IFP appeared to precede the changes observed in functional MVD (Fig. 4). Doubling the sHHI dose did not reduce IFP further (Supplementary Fig. S6).

Although SMO antagonists have been reported to mediate stromal thinning in PDAC, it is unclear as to how desmoplastic components respond dynamically to treatment. Therefore, effects upon the ECM components collagen I and HA were investigated. Collagen I expression is abundant in PDAC, contributing to the drug delivery barrier and also playing a role in metastasis and drug resistance (7, 8, 11, 42, 43). Whereas the collagen I network appeared as extended, thick, fiber-like structures in stromal regions of control tumors, fibers were thinner and shorter after 7 days of sHHI treatment (Fig. 3B). Within 3 days of initiating sHHI treatment, collagen I decreased significantly (P ≤ 0.05) and reached a nadir on day 8 (Fig. 3C).

The HA network constitutes a more dynamic component of the tumor ECM and is associated with high interstitial pressures in PDAC (6). Hyaluronic acid synthase 2, a key enzyme in HA synthesis, is regulated by the sHH signaling pathway (16). Daily sHHI treatment reduced HA abundance significantly within 3 days (Fig. 3D and E; P ≤ 0.05), and it reached a nadir after 8 to 10 days. In particular, the regions of greatest HA abundance became less prevalent with sHHI treatment (Fig. 3E). Variability in the data increased after 10 to 20 days of continuous dosing, suggesting possible onset of sHHI tolerance. Temporally, the changes in HA and collagen 1 paralleled the decrease in tumor IFP, which appeared to coincide with reperfusion of the tumor microvasculature (Fig. 4).

The data together suggested that sHHI effects are dose- and time-dependent, and result in: (i) increased perfusion of the tumor via an increase in fraction of functional microvessels, (ii) reduced density of the stromal matrix, and (iii) reduction in tumor IFP. Therefore, we investigated whether these changes correlate with changes in tumor penetration and deposition of therapeutic mAb. Cetuximab was selected as the proof-of-concept targeted mAb, owing to the prevalence of EGFR overexpression in PDAC. A nontargeted control mAb, 8C2 (anti-topotecan), was used to probe nonspecific mAb deposition.

PDX tumor #18269 expresses EGFR and showed some degree of EGFR signaling in tumor cells, based upon total- and phospho-EGFR expression (Supplementary Fig. S7A and S7B). The targeted and nontargeted mAb were labeled with complementary, hydrophilic fluorophores, mixed, and coadministered intravenously on day 7 of a 13-day sHHI dosing regimen, which was designed to coincide with the time of peak functional MVD and the nadir of IFP, HA, and collagen I (Fig. 4). Little tumor mAb deposition was observed without sHHI priming (Fig. 5A). In contrast, cetuximab fluorescence was intense in sHHI-primed animals 12 hours after mAb administration, and localized primarily with tumor cells at the stromal border. Deposition of nontargeted mAb 8C2 also increased with sHHI priming, but it was distributed throughout the tumor (Supplementary Fig. S8).

The kinetics of mAb deposition and distribution were analyzed by serial sampling of control- and sHHI-pretreated mice over 7 days. Tissue images were segmented into tumor and stromal regions, and mAb fluorescence intensity was quantified. The kinetics of cetuximab tumor deposition in primed and nonprimed animals were similar, but the area under the concentration–time curves (AUC) differed markedly. In both groups, cetuximab deposition in tumor cell regions peaked 12 hours after injection (Fig. 5B) but more than doubled in sHHI-primed mice. The similarity of cetuximab kinetics in primed versus nonprimed tumors, but with differences in magnitude, suggests that sHHI priming increased the tumor cell compartment accessible to mAb circulating in plasma. Deposition of the nontargeted mAb also increased with sHHI priming, but the kinetics differed from nonprimed animals. With sHHI pretreatment, 8C3 concentrations showed a rapid rise (Fig. 5C), with a temporal profile similar to that of cetuximab in sHHI-primed animals, and its tumor AUC nearly doubled. In nonprimed animals, 8C2 tumor concentrations increased slowly, reaching peak ≥150 hours after administration. The results together demonstrate that sHHI pretreatment modulates tumor architecture so as to increase the volume of tumor that is accessible to circulating mAb, and the enhancement in deposition is not mAb-dependent.

Given that sHHI increased mAb access to tumor cells, we investigated the therapeutic efficacy of the sHHI/cetuximab combination. Rituximab (anti-CD20) was used as a species-matched, nontargeted control mAb. Based upon the observed temporal changes in tumor and stroma, a 14-day treatment cycle was designed, in which the sHHI was administered for the first 10 days, and withdrawn for the last 4 days (Supplementary Fig. S9). The purpose of the drug holiday was to reduce the potential for pharmacodynamic tolerance to the sHHI priming effect, which was reported to occur within 2 weeks of treatment (33), and to avoid potential adverse effects of continuous stromal inhibition (24–26). Cetuximab pharmacokinetics were analyzed using a two-compartment model (Supplementary Fig. S10A and S10B; Supplementary Table S2). With consideration of the temporal characteristics of tumor priming, modeling and simulation supported mAb administration on the 5th, 8th, and 11th days of each 14-day cycle. Despite the high plasma concentrations of cetuximab achieved by this dosing regimen (>10 μg/mL; Fig. 6A), cetuximab had no effect upon tumor volume progression in nonprimed animals (Fig. 6A; Supplementary Fig. S11). In striking contrast, sHHI pretreatment combined with cetuximab retarded tumor volume progression as treatment cycles continued. Rituximab with sHHI was no more effective than the vehicle control. Tumor volume in sHHI-primed animals treated with cetuximab followed an undulating pattern that became apparent after the third treatment cycle. Upon withdrawal of the sHHI, tumor volume rebounded, despite the fact that the 3rd cetuximab injection of each cycle resulted in the highest plasma mAb concentrations (Fig. 6A). Resumption of sHHI dosing after the drug holiday resulted in an immediate decline in tumor volume that was not observed in the absence of coadministered cetuximab.

Only sHHI alone and combined sHHI/cetuximab significantly extended survival to a tumor volume limit (TVL) of 2,000 mm³ (Fig. 6B). For most groups (vehicle control, cetuximab alone, rituximab ± sHHI), median survival to TVL was approximately 43 days. For the sHHI alone, median survival to TVL was 67 days. Median survival to TVL for the sHHI/cetuximab regimen was 101 days, 2.4-fold greater than controls. This increase was significant compared with controls (P ≤ 0.01) or animals treated with sHHI alone (P ≤ 0.05).

PDAC is a frequently lethal cancer for which chemotherapy is seldom successful, and therapeutic antibodies have had little impact upon PDAC outcome (2, 44). The numerous mechanisms of therapy resistance or failure are incompletely understood; contributing pathophysiologic and molecular mechanisms include mutations in signaling pathways for growth and survival, extensive desmoplasia, and low MVD, perfusion, and permeability (4, 6, 12, 13, 45, 46). For macromolecular agents, the dense stroma, elevated IFP, and tissue solid stress constitute key barriers interposed between the systemic circulation and delivery to tumor cells (4–7, 9, 10, 45, 47). Hypovascularity hinders convective delivery of mAb to the tumor, elevated IFP may cause an outward convective flow opposing delivery, tissue solid stress impedes interstitial diffusion of mAb after extravasation, and together, solid stress and high IFP reduce perfusion by collapsing functional microvasculature. Any strategy to enhance therapeutic antibody delivery to PDAC cells must address these barriers concurrently.

Tumor-priming strategies aim to compromise the drug delivery barrier and enhance deposition of subsequently administered agent(s), and the priming approach investigated here involves interdiction of sHH signaling, which plays an important role in desmoplasia. Inhibitors of this signaling axis compromise the barriers to drug delivery by: (i) increasing tumor vascularity, permeability, and/or perfusion, (ii) reducing tumor IFP and stromal integrity to reduce tissue solid stress, and (iii) increasing intratumor diffusion (4, 9, 16, 17, 19, 22–24). Multiple PDAC PDX tumors responded to sHH inhibitors with a deep reduction in Gli1 expression and increased permeability to nanoparticulate perfusion/permeability probes. One PDX model was selected for more comprehensive investigation of sHHI effects upon mAb deposition and intratumor distribution based upon its hypovascularity, poor permeability and perfusion, extensive desmoplasia, and detectable EGFR signaling activity. The pharmacodynamics of changes in tumor architecture and vasculature were investigated for two sHHI dose levels. Short-term treatment at a lower dose (40 mg/kg/day) increased functional vessel density significantly, but total vessel density did not increase. A higher dose (80 mg/kg/day) resulted in rapid but more transient changes in functional vascularity, suggesting the importance of optimizing dose and regimen. The sHHI treatment decreased the density of the ECM constituents HA and collagen I in parallel with the reduction in IFP, relieving compression of existing vasculature, restoring perfusion, and enhancing convective delivery of antibody drugs (Fig. 4). Hydration of HA and increased collagen I elevate IFP and tissue solid stress, which consequently collapses tumor vessels (6–8). Responses of collagen I and HA to sHHI treatment were time-dependent and suggest that the priming window for enhanced tumor cell exposure to antibody also may be transient.

The desmoplastic stroma and high intratumor pressures impede mAb delivery following extravasation, resulting in spatially heterogeneous intratumor distribution (12). Typically, mAb concentrations fall below a therapeutic threshold in regions distal to the tumor vasculature because of delivery barriers, thereby compromising therapeutic efficacy and potentially accelerating the emergence of drug resistance (13). Priming increased the exposure of tumor cells to convective delivery, the amount of mAb deposited, and the depth of penetration.

We hypothesize that short-term exposure to SMO antagonists, interspersed with drug-free holidays, may accomplish tumor priming while minimizing onset of pharmacodynamic tolerance (33) and deleterious effects of long-term inhibition. Recent reports demonstrate that reduction in desmoplasia, or genetic ablation of sHH signaling or tumor-associated fibroblasts, promotes a more malignant, metastatic tumor phenotype (24–26). Concerns raised in an initial small-scale clinical trial of combined gemcitabine/sHHI resulted in suspension of similar trials (48). However, additional trials showed no evidence of inferiority or toxicity of the SMO antagonist treatment arms (49). Based upon pharmacodynamic analysis, 5- to 10-day sHHI treatment was selected here as the tumor-priming regimen for subsequent administration of cetuximab. Estimates of tumor EGFR expression (50) suggested that a dose of 7 mg/kg cetuximab would constitute a saturating dose sufficient to block all accessible tumor EGFR. The kinetic data for fluorescent mAb deposition supported this prediction; the majority of the tumor bulk was not accessible to cetuximab in treatment-naïve (nonprimed) animals, whereas 7 days of sHHI pretreatment rendered the tumor interstitium more accessible to mAb. In primed versus nonprimed animals, the kinetics of cetuximab tumor deposition were parallel, but the magnitude of deposition differed. The results suggest that tumor cell EGFR that is accessible to mAb saturates rapidly with and without priming, and that the effect of sHHI pretreatment is to increase the number of tumor cells accessible to circulating mAb.

The biodistributional effects of sHHI tumor priming upon cetuximab disposition increased therapeutic efficacy. Unsurprisingly, cetuximab in the absence of priming had no therapeutic effect in this EGFR-expressing PDX model, just as cetuximab lacks clinical activity in PDAC (32). However, repeated cycles of sHHI priming and cetuximab treatment arrested mean tumor volume progression in a fashion that was clearly dependent upon treatment with both agents. Three initial treatment cycles did not inhibit tumor volume progression perceptibly. However, in subsequent cycles, average tumor volume did not progress, despite oscillations around a mean that clearly reflected the initiation and cessation of each treatment cycle. These tumor volume oscillations represent an interesting phenomenon of undetermined underlying mechanism(s). Contributing factors likely include the timing of sHHI and mAb administration and the sHHI drug holiday. The contribution of EGFR target engagement is unclear in light of KRAS oncogenic signaling. Other possible contributions include antibody-dependent cell-mediated cytotoxicity, given that SCID mice have natural killer cells, neutrophils, and macrophages, and are able to respond to cetuximab (51).

Because intratumor mAb concentrations are far lower than required to saturate tumor EGFR, it would be important to maintain high mAb plasma concentrations during both the priming window and the drug holiday to maximize delivery. Previous studies showed that 3 days after cessation of a 10-day sHHI dosing regimen, tumor permeability to 80 nm nanoparticles remained elevated (22). Therefore, the day 11 cetuximab dose, administered during the drug holiday, also would have access to the tumor interstitium. For most PDX models, priming effects appeared by approximately day 4, and the earliest suggestion of sHHI pharmacodynamic tolerance was observed at ≥day 10 of dosing; pharmacodynamic tolerance to NVP-LDE225 was reported to occur in vitro in as few as 13 days (33). A local nadir in tumor volume was observed mid-cycle in each of the last three treatment cycles. The rapid reinduction of tumor volume suppression upon reinitiation of sHHI treatment suggests that the 4-day drug holiday restored tumor sensitivity to sHHI treatment effects. Thus, a sequence of low sHHI doses, administered for the shortest interval necessary to mediate tumor priming, and interspersed with a drug holiday, may underlie success of this combination therapy strategy. The priming strategy would not be limited to cetuximab, as the data show that tumor deposition of a nontargeted, control mAb also increased during sHHI priming. These hypotheses are under investigation.

No potential conflicts of interest were disclosed.

Conception and design: J. Wang, A. Sen, W.W. Ma, R.M. Straubinger

Development of methodology: J. Wang, W.W. Ma, R.M. Straubinger

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J. Wang, D.K.W. Chan, A. Sen

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J. Wang, D.K.W. Chan, A. Sen, W.W. Ma, R.M. Straubinger

Writing, review, and/or revision of the manuscript: J. Wang, A. Sen, W.W. Ma, R.M. Straubinger

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): D.K.W. Chan

Study supervision: W.W. Ma, R.M. Straubinger

We thank Drs. B.L. Hylander and E.A. Repasky for advice and access to the PDAC tumor panel, R.F. Pitoniak, MSN, for curating the tumor panel, Dr. T. Roy Chaudhuri for helpful discussions, and Y. Qu and N.L. Straubinger for technical assistance. We thank Drs. L. Luus, W. Kamoun, and D.C. Drummond (Merrimack Pharmaceuticals Inc.) for sharing their unpublished MVD measurements and preliminary mutation analysis for these tumors. Novartis provided some of the NVP-LDE225.

Support for this work was provided by NIH/NCI grants R21CA168454 and R01CA198096 (R.M. Straubinger and W.W. Ma), and a grant from the UB/SUNY Center for Protein Therapeutics consortium (R.M. Straubinger). Comprehensive Cancer Center Support grant NIH/NCI P30CA016056 to Roswell Park Comprehensive Cancer Center provided shared resources utilized in the work. J. Wang received partial support from an unrestricted predoctoral fellowship provided to the UB Department of Pharmaceutical Sciences by Genentech, Inc. A. Sen also received support from NIH/NCI grant R21CA194634.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.