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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Int J Cancer. Author manuscript; available in PMC 2017 April 1.
Published in final edited form as:
PMCID: PMC4724303
NIHMSID: NIHMS731128

Perioperative treatment with the new synthetic TLR-4 agonist GLA-SE reduces cancer metastasis without adverse effects

Abstract

The use of TLR agonists as an anti-cancer treatment is gaining momentum given their capacity to activate various host cellular responses through the secretion of inflammatory cytokines and type-I interferons. It is now also recognized that the perioperative period is a window of opportunity for various interventions aiming at reducing the risk of cancer metastases – the major cause of cancer related death. However, immune-stimulatory approach has not been used perioperatively given several contraindications to surgery. To overcome these obstacles, in the current study we employed the newly introduced, fully synthetic TLR-4 agonist, Glucopyranosyl Lipid-A (GLA-SE), in various models of cancer metastases, and in the context of acute stress or surgery. Without exerting evident adverse effects, a single systemic administration of GLA-SE rapidly and dose dependently elevated both innate and adaptive immunity in the circulation, lungs, and the lymphatic system. Importantly, GLA-SE treatment led to reduced metastatic development of a mammary adenocarcinoma and a colon carcinoma by approximately 40-75% in F344 rats and BALB/c mice, respectively, at least partly through elevating marginating-pulmonary NK cell cytotoxicity. GLA-SE is safe and well tolerated in humans, and currently is used as an adjuvant in phase-II clinical trials. Given that the TLR-4 receptor and its signaling cascade is highly conserved throughout evolution, our current results suggest that GLA-SE may be a promising immune stimulatory agent in the context of oncological surgeries, aiming to reduce long-term cancer recurrence.

Keywords: TLR-4, metastases, in vivo, NK, stress, surgery, MADB106, CT26, perioperative, GLA

Introduction

Cells of the innate immune system, including dendritic cells (DC), macrophages, natural killer cells (NK), and monocytes, identify potential threats by employing numerous receptor types1,2, among them are the toll-like receptors (TLR) family3. These transmembranal receptors are highly conserved throughout evolution, and recognize pathogen-associated molecular patterns (PAMPs) derived from viruses, bacteria, fungi, and parasitic-protozoa. Thirteen mammalian TLRs have been recognized thus far, 10 of which are also expressed in humans (TLRs 1-10), and 3 which are solely expressed in mice (TLRs 11-13)4. All of the TLRs induce a signal transduction through either the adapter protein MyD88 or TRIF. This downstream activation leads to the secretion of inflammatory cytokines and type-I interferons, which results in various host cellular responses, including proliferation and a robust adaptive and innate immune activation5.

These TLR-derived cellular responses have prompted researchers to examine the use of TLR agonists as anti-cancer treatments. Such treatments rely on TLR's ability to initiate T cell activation; DC maturation; DC antigen uptake, processing, and presentation; enhancement of NK and CTL cytotoxicity, and other immune enhancing responses6,7. In fact, TLRs 3, 4, 7, 8, and 9 agonists have shown promising therapeutic potential in the field of cancer, and are included in the US NCI list of agents with highest potential for cancer treatment7,8.

The use of immune stimulatory agents, including TLR agonists, has not been studied in the immediate context of surgical excision of a primary tumor, given their common pro-inflammatory adverse impacts, which are contra-indicated to surgery9. However, the perioperative period is now recognized as a window of opportunity for reducing the risk for long-term cancer metastases10-12 - the major cause of cancer related death13,14. Consequently, various interventions, including immune stimulation employing TLR agonists, should be adapted to the perioperative period in an attempt to improve long-term cancer outcomes12.

TLR-4 was the first TLR discovered in humans (1998)15. It recognizes lipopolysaccharide (LPS), an endotoxin found on the outer membrane of Gram-negative bacteria. This recognition is mediated by the co-receptor MD-2, which binds the endotoxin and heterodimerizes with TLR-4, allowing the activation of either the MyD88 or the TRIF dependent pathway16. Following this activation, antigen presenting cells (APCs) are stimulated to produce and secrete pro-inflammatory cytokines, including IFN-α, which further promote the proliferation of CD4+ T cells, as well as the production of IFN-γ, TNF-α, perforin and granzyme by CD8+ cytotoxic T cells (CTLs) and NK cells6,17,18.

LPS has been used as an immune-stimulator in vitro and in vivo long before the mechanisms of its actions were revealed. Apart from its positive stimulatory effects, LPS has been shown to cause various adverse effects. It can lead to an exaggerated pyrogenic reaction, and at high doses may cause septic shock19. Furthermore, we have previously shown that low doses of LPS, injected simultaneously or shortly before tumor inoculation, resulted in higher organ retention level of tumor cells20. These deleterious effects were attenuated through the blockade of inflammatory or sympathetic responses20. To overcome the many adverse effects of LPS, modified variations have been introduced, each with unique characteristics and different TLR-4 activation potential, including biological derivatives of the original LPS molecule, such as the monophosphoryl lipid A (MPL)21, as well as fully synthetic substitutes.

Glucopyranosyl Lipid-A, administered within a stable emulsion (GLA-SE), is a newly introduced synthetic TLR-4 agonist, which has been used to date, in various phase-I/II clinical trials in the USA in more than 1,000 subjects with no significant adverse effects22. Unlike naturally occurring endotoxins and biological derivatives, GLA has a defined number and length of carbon chains, and lacks sugar residues on a specific hydroxyl connection site21 (Sup. Fig. 1). This unique structure leads to a highly efficient Th1 reaction, but, unlike LPS and other natural endotoxins, minimizes the Th2 response, thus reducing adverse effects. For example, comparing RNA levels in mice following LPS or GLA administration, similar expression levels of TNF-α and IL-12 were evident, while GLA led to lower expression levels of IL-6, and higher expression levels of the NK-activating IL-1521. GLA-SE has not been studied thus far in the context of either cancer or surgery, but its aforementioned characteristics present a unique potential for perioperative use in an attempt to arrest the metastatic process.

In the current study we examined GLA-SE therapeutic use employing various models of cancer metastases. We aimed at finding an optimal regimen of GLA-SE, which will yield anti-metastatic effects with minimal adverse effects. We further aimed at elucidating the immunological mechanisms of its action in the context of cancer metastasis. Outcomes were often examined in the context of acute stress or surgery, including a β-adrenergic challenge, to better mimic the clinical perioperative settings23,24. The results indicated that a single systemic administration of GLA-SE rapidly and dose dependently elevated both innate and adaptive immunity in various compartments, with no evident adverse effects, and led to reduced metastatic development in all examined tumor models.

Materials & Methods

1. Animals

Male and female Fischer 344 (F344) rats, 3-6 months old, and 8 weeks old male BALB/c mice, were housed 3–4 per cage in our vivarium with ad-libidum access to food and water on a 12:12 light–dark cycle at 22 ± 1 °C. Rats were handled 4 times prior to experimentation to reduce potential procedural stress. Age, weight, sex, and drug administration were counterbalanced across all experimental procedures. Housing conditions were monitored by the Institutional Animal Care and Use Committee of Tel Aviv University, which also approved all studies described herein.

2. Drugs

2.1 Glucopyranosyl Lipid-A SE (GLA-SE) and its administration

GLA (IDRI, Seattle, WA), a synthetic TLR-4 agonist21, dissolved in a stable emulsion (SE, see below, 2000 μg GLA in 1ml SE) was further diluted in SE or PBS for a final concentration of 3μg/ml-200μg/ml.

2.2 Stable emulsion (SE) delivery system for GLA

SE (IDRI, Seattle, WA) is an oil-in-water emulsion manufactured by high-shear homogenization. The oil droplets are stabilized by emulsifiers in a bulk aqueous phase, and serve as an adjuvant delivery system25. Since GLA is dissolved in SE (hence, GLA-SE), SE served as a control condition in some of the experiments conducted. In these cases, SE was diluted and administered in the exact manner as GLA-SE.

2.3 Polyriboinosinic acid (Poly I-C)

Poly I-C (Sigma, Israel) is a synthetic compound that resembles viral double-stranded RNA and triggers an anti-viral immune response26. Poly I-C has been widely used as an immune stimulator27-29 inducing reliable inflammatory NK activating responses28,30.

2.4 Anti-NKR-P1

Anti-CD161 binds to the NKR-P1 surface antigen expressed on NK cells in rats, and, to a much lesser degree, on rats polymorphonuclear (PMNs) cells. In vivo treatment of rats with anti-NKR-P1 selectively depletes NK cells and eliminates NK- and antibody-dependent non-MHC-restricted cell cytotoxicity. T cell function and the percentages of T cells, PBMCs, and PMNs are unaffected. This antibody renders NK cells ineffective in vivo immediately upon administration, and selectively depletes NK cells within a day31. 1.5mg/kg of the antibody is injected i.v. simultaneously with MADB106 tumor cell inoculation.

3. Tumor cell lines

3.1 MADB106

MADB106 is a selected variant cell line obtained from a pulmonary metastasis of a chemically induced mammary adenocarcinoma (MADB100) in the F344 rat. Following i.v. inoculation, MADB106 tumor cells metastasize only to the lungs32, and lung tumor retention (LTR), which is highly indicative of the number of metastases that would have developed weeks later, is dependent upon NK cells in this model33-35. For in vitro maintenance and harvesting of MADB106 cells See36. This cell line was used for both in vivo assessment of lung tumor retention and lung metastases, and in vitro examination of NK cell cytotoxicity (NKCC).

3.2 YAC-1

YAC-1 lymphoma is the standard target cell line used for the assessment of rodent in vitro NK cytotoxicity. The cell line was maintained in suspension cultures in CM in 100% humidity, 5% CO2 at 37 °C. This cell line was used for in vitro examination of NK cytotoxicity.

3.3 CT26

The CT26 murine colon carcinoma cell line is a chemically induced undifferentiated carcinoma, syngeneic to the BALB/c strain37. For in vitro maintenance and harvesting of CT26 cells See38. This cell line was used for in vivo assessment of hepatic metastases.

4. Radiolabeling of MADB106 tumor cells and assessment of lung tumor retention (LTR)

Tumor cell DNA radiolabeling for assessment of LTR is accomplished by adding 0.5 μCi/ml of 125Iododeoxyuridine (Perkin-Elmer, Israel) to the cell culture for 24h. For i.v. tumor cell injection, rats are lightly anesthetized with isoflurane, and 4 × 105/kg MADB106 tumor cells in 0.5ml PBS containing 0.1% BSA were administered. For assessment of LTR, animals were sacrificed with an overdose of CO2 24h after tumor inoculation, and lungs were removed to assess retained radioactivity. LTR calculation: (radioactivity count of lung – background radioactivity) × 100/(radioactivity count of the total injected cell suspension – background radioactivity), hence, lower LTR results indicate better clearance of tumor cells from the lungs.

5. The use of adrenergic challenge simultaneously with MADB106 inoculation

LTR was often tested in the context of pharmacological stress induced simultaneously with tumor inoculation by administering epinephrine dissolved in a slow-release emulsion36. This adrenergic challenge is used for two reasons. First, it often characterizes the stressful clinical setting where immune-stimulatory approaches are employed or could be beneficial (e.g., having cancer or undergoing surgical removal of a primary tumor), and studies showed the metastasis promoting effects of catecholamines (34-48-49). Second, we previously found that under such adrenergic challenge the beneficial effects of immune stimulation are better reflected36,39.

6. Lung metastases model

Non-labeled MADB106 tumor cells (105/animal in 0.5ml PBS supplemented with 0.1% BSA) were injected i.v. to lightly anesthetized rats. Three weeks later, all animals were sacrificed and their lungs were removed and placed in Bouin's solution39 for 24hrs. Lungs were then washed in ethanol prior to systematic enumeration of visible extra-pulmonary metastases by an experimenter unaware of the origin of each lung.

7. Colon carcinoma model

Mice were anesthetized and maintained with 1.5-2.5% isoflurane. The skin was shaved and rubbed with ethanol pads, and a 0.5-cm abdominal incision was made adjacent to the spleen. 1×104 CT26 tumor cells were injected into the spleen using a 31G needle. A 4/0 blue polypropylene monofilament non-absorbable suture was placed across the hilum of the spleen to prevent bleeding, and a splenectomy was performed. Thereafter, the peritoneum and skin were sutured with 4/0 non-absorbable filaments38. Twenty days following tumor inoculation animals were euthanized with isoflurane, livers were harvested and weighted, and surface hepatic metastases were counted by an investigator blinded to each animal’s experimental group.

8. Harvesting and preparing circulating and marginating-pulmonary (MP) leukocytes

Rats were sacrificed with isoflurane and the peritoneal and chest cavities opened. Blood was collected from the right ventricle of the heart into heparinized syringes. One ml of blood was washed once with 3 ml of PBS (400g for 10min) and twice with 3 ml of CM, and reconstituted to its original volume. The remaining blood was centrifuged (931g in 4°C for 20min) and plasma was collected and stored in −32°C for ELISA. MP leukocytes were harvested by perfusing the lungs through the heart as described in details elsewhere39.

9. Harvesting and preparing extracted lymph nodes

Rats were sacrificed with isoflurane and the peritoneal skin was separated from the muscle and pulled sideways. One to three (changed between experiments) inguinal lymph nodes from each side of the rat were removed intact using a curved-end tweezers. Each node was homogenized using 300μl PBS through a 40μm cell strainer, followed by centrifugation (670g for 10min). Supernatant was collected and stored in −32°C for ELISA, and the pellet was re-suspended in 50μl PBS++ for flow cytometry analysis.

10. Assessment of NK cytotoxicity

The standard whole blood 51Cr release assay was used. Six different effector-to-target (E:T) ratios were formed by serially diluting 150μl aliquots of the effector-cell preparation in a microtiter plate. Then, 5000 radiolabeled target cells (MADB106 or YAC-1) in 100μl CM were added to each well on top of the effector-cell preparation. Spontaneous and maximal releases of radioactivity were determined by substituting effector cells with CM or Triton-X100 (Sigma, Rehovot, Israel), respectively. For more details see Supplementary Methods.

11. Flow cytometry

11.1 Sample preparation

A 50μl aliquot of whole blood, lung perfusate, or suspended lymph nodes was added to 50μl of PBS supplemented with 2% FCS and 0.1% NaN3 (PBS++) containing a set of conjugated antibodies. Samples were incubated for 15min in the dark at room temperature, and 1ml FACS lysing solution (Becton Dickinson) was added. Twelve min later, samples were washed twice with 1ml PBS++ (670g for 5min) and resuspended in 300μl of PBS++ for flow cytometry, using a FACScan (Becton Dickinson). To assess the absolute number of cells, or a specific cell subtype, per μl of sample, 300 polystyrene microbeads (20μm, Duke Scientific, Palo Alto) per μl sample were added to each sample, and the following formula was used: (#cells/#microbeads)×300.

11.2 Identification of leukocytes subsets and cellular expression markers

Granulocytes and lymphocytes were identified based on forward by side scatter. Within lymphocytes, monocytes were identified by the APC-conjugated anti-CD161 mAb (Biolegend, San Diego, CA) as being CD161dim cells, and NK cells were identified by as being CD161bright cells. This criterion has been shown to exclusively identify more than 95% of cells that exhibit NK activity40. T cells were identified using a PerCP-eFluor710-conjugated anti-CD3 mAb (eBioscience, San Diego). Expression of activation markers was analyzed using the FITC-conjugated anti-FasL mAb (Acris, Herford, Germany) and the PE-conjugate anti-NKG2D mAb (eBiosceince, San Diego, CA).

12. Cytokines assessment using ELISA

The anti-rat IL-1β, IL-6, IL-12, IL-15, and IFN-γ (PeproTech, Rocky Hill, NJ) were used to assess cytokine levels from plasma and lymph supernatant, according to the manufacturer’s instructions.

13. Assessment of social exploratory behavior

Male adult rats were habituated to the examination cage for 30min, 24h prior to the actual assessment, during the dark phase and under red light illumination. In the examination day, each rat was placed in the examination cage, and was allowed 5min for habituation, following which a juvenile male rat (23–29 days old) was introduced. Each social exploration session was video recorded for offline analysis.

Social exploration was defined as engaging by the adult rat in anogenital sniffing, and/or intent close pursuit of the juvenile. Aggressive behaviors such as biting and mounting were rarely observed. The total duration of social exploration was assessed offline along the first 10min period of the session by a researcher blind to the treatment of the observed adult rat.

14. Statistical analyses

One, two-way or repeated measures analysis of variance (ANOVA) with a pre-determined significance level of 0.05 was conducted. Provided significant group differences were found, Fisher's protected least significant differences (Fisher's PLSD) contrasts were performed to compare specific pairs of groups, based on a priori hypotheses that GLA-SE improves immunological indices and resistance to metastasis. Pearson correlations were used for assessing the associations between average NK cytotoxicity levels and number of NK cells.

Procedures and Results

GLA-SE dose-dependently reduces MADB106 lung tumor retention (LTR)

Procedure

Seventy-five 3-months old F344 male rats were randomly assigned to one of 7 treatments, and administered s.c. with 100μl of GLA-SE in a dose of 0.3μg, 0.7μg, 2.5μg, 10μg, or 20μg/animal, or with control injection of PBS or SE. Twenty-four hours later, MADB106 cells were inoculated, half of each group received epinephrine (see methods), in order to simulate stress/surgery conditions along with tumor inoculation, and the other half received vehicle. Twenty-four hours later, lungs were removed for assessment of LTR.

Results

GLA-SE significantly reduced MADB106 LTR dose dependently in both the epinephrine and vehicle groups (ANOVA: F(6,31)=14.375, p<0.0001; F(6,30)=3.354, p<0.05, respectively). SE alone reduced LTR only under epinephrine conditions (p<0.001), and GLA-SE further reduced LTR beyond SE alone in the epinephrine condition in the doses of 0.7μg, 2.5μg, 10μg, and 20μg (p<0.05 to p<0.001), and without epinephrine in the doses of 2.5μg and 20μg (p<0.05 to p<0.001) (Fig. 1A/B).

Figure 1
GLA-SE dose-dependently improves lung tumor retention, both in the context of adrenergic challenge (A) and without it (B). * indicates a significant difference from PBS at p<0.05; ** indicates a significant difference from PBS at p<0.0001; ...

An intermediate but effective dose of 2μg/animal GLA-SE was used in the following studies as our standard treatment. Additionally, as SE alone reduced LTR, we examined the effects of diluting GLA-SE in PBS and found no reduction in its efficacy (see supplemental data, and Sup. Fig. 2).

Subcutaneous, but not intraperitoneal administration, is the effective delivery method for GLA-SE

Procedure

Fifty-one 4-months old F344 male rats received either intraperitoneal or subcutaneous injection of either PBS, SE, or GLA-SE (2μg/animal in 100μl). 24h later, MADB106 cells were inoculated simultaneously with epinephrine administration. 24h later, animals were sacrificed and lungs removed for LTR assessment.

Results

Only the subcutaneous delivery approach reduced LTR (Sup. Fig. 3), as indicated by a significant interaction between treatment and delivery method (F(2,45)=3.937, p<0.05). Fisher’s PLSD post-hoc comparisons yielded a significant difference between i.p. and s.c. (p<0.01)

The beneficial effects of GLA-SE on LTR are evident as early as 4h following administration, with faster response time in males

Time course of the beneficial effects of GLA-SE was examined in both sexes.

Procedure

Male F344 rats (n=75, 6-months old) were injected with either PBS, SE, or GLA-SE (2μg/animal, s.c. 100μl) at 0h, 4h, 12h, 24h, or 48h prior to tumor inoculation. All animals received epinephrine with MADB106 cells, and 24h later lungs were removed for LTR assessment. The same experimental design was conducted in females (n=89), with the addition of a 96h time point.

Results

In both sexes, no differences were evident between the different times of PBS or SE administration, and data in each of these control conditions was combined for analysis.

ANOVA indicated time dependent effects in both males and females (F(6,67)=5.695, p<0.0001; F(7,81)=2.489 , p<0.05, respectively). In males, GLA-SE reduced LTR at all time points (4h, 12h, 24h, 48h; p<0.05-0.001), and in females at the 24h and 48h (p<0.05) (Sup. Fig. 4A/B).

These results indicate a quick response with a long lasting effect for a single intermediate dose injection of GLA-SE in both sexes.

GLA-SE administration does not cause behavioral or physiological adverse effects

Procedure

Seventeen 3-months old F344 male rats received either PBS, GLA-SE (2μg/animal), or a standard dose of the TLR-3 agonist Poly I:C (4mg/kg) (s.c. 100μl). 24h later all animals underwent the social exploration test. Each animal was weighted daily for 4 days, beginning on injection day.

Results

Poly I:C, but not GLA-SE, reduced exploration behavior (p<0.05) (ANOVA: F(1,14)=4.105, p<0.05) (Fig. 2A). Similarly, poly I:C, but not GLA-SE, reduced body weight (p<0.001) (Repeated measures ANOVA: F(2,14)=13.782, p<0.001) (Fig. 2B).

Figure 2
No adverse effects were evident following GLA-SE administration (2μg/animal), in contrast to behavioral and physiological adverse effects of Poly I:C, as indicated by weight loss (A) and reduced social interaction time (B). * indicates a significant ...

These results indicate no behavioral or physiological adverse effects for GLA-SE treatment.

The beneficial effects of GLA-SE on LTR are mainly mediated through activation of NK cells

The beneficial effects of GLA-SE could be mediated through various mechanisms. To test the possible mediating role of NK cells, we compared the effects of GLA-SE between naïve rats and rats selectively depleted of NK cells.

Procedure

Fifty-four 4-month old F344 male rats received either PBS, SE, or GLA-SE (2μg/animal) (s.c. 100μl). 24h later, MADB106 cells were administered simultaneously with either the anti-NKR-P1 mAb or with vehicle. 24h later, lungs were removed for LTR assessment. No adrenergic challenge was added due to known direct effects of epinephrine on NK, and potential different reaction to epinephrine in the NK-depleted group, which might have obscured and confounded the results.

Given an expected 100-fold difference in LTR levels (and in variance) caused by depletion, data was calculated and presented as percent of the respective control (PBS) for each group.

Results

The beneficial effects of GLA-SE were evident only in naïve (p<0.001), but not in NK-depleted rats, as indicated by a significant interaction between treatment and depletion (ANOVA: F(2,46)=7.354, p<0.01) (Fig. 3).

Figure 3
The beneficial effects of GLA-SE (2μg/animal) are mediated through NK cells, as its effects completely vanished in their absence. * indicates a significant difference from PBS at p<0.001. LTR is shown as % of relevant PBS control. Data ...

Time dependent changes in circulating and marginating-pulmonary NK cell numbers, NK cytotoxicity, and cytokine secretion, following administration of GLA-SE

In the clinical practice it would be beneficial to know the kinetics of the immune stimulatory effects of GLA-SE in order to optimize its benefits.

Procedure

Fifty-two 4-months old F344 male rats received PBS (s.c. 100μl), 2μg/animal GLA-SE at 4h, 12h, or 24h before sample collection, or 8μg/animal GLA-SE 24h before sample collection. At time 0h, blood and marginating pulmonary leukocytes were harvested for the assessment of NK cytotoxicity, flow cytometry, and ELISA (see methods).

Results:

Circulation

NK cell numbers, cytokine levels, and cytotoxicity against YAC-1 target cells – GLA-SE lowered NK cell numbers/μL blood at the 4h and 12h time-points (p<0.01) (ANOVA: (F(4,46)=3.304, p<0.05), which returned to baseline at the 24h time-points (Sup. Fig. 5A). Cytotoxicity levels/μL blood showed a similar pattern: decreasing at the 4h time-point (p<0.01), gradually returning to baseline levels at 12h and 24h, but, unlike the numbers, exceeding baseline levels in the 24h 8μg group (p<0.05) (Repeated measures ANOVA: F(4,47)=6.581, p<0.001) (Fig. 4A). IL-15 and IFN-γ levels were elevated at 24h (p<0.01) (ANOVA: F(4,45)=8.492, p<0.0001, and F(4,39)=5.137, p<0.01, respectively) (Sup. Fig. 7A/B).

Figure 4
Circulating NK cytotoxicity per ml blood against YAC-1 target cells was lowered 4 and 12h following GLA-SE administration and returned to baseline at 24h (A). In contrast, no decrease in marginating-pulmonary NK (MP-NK) cytotoxicity was evident, and an ...

Marginating-pulmonary leukocytes: Numbers and cytotoxicity of MP-NK cells against YAC-1 and MADB106 target cells – 2μg/animal GLA-SE elevated MP-NK cell numbers at 12h (p<0.01), which returned to baseline levels thereafter. An elevation in MP-NK numbers was also evident in the 8μg 24h (p<0.05) (ANOVA: F(4,47)=3.174, p<0.05) (Sup. Fig. 5B). GLA-SE elevated cytotoxicity levels against YAC-1 at 12h and 24h, and against MADB106 at 24h (p<0.05 for all) (Repeated measures ANOVA: YAC-1- F(4,47)=7.883, p<0.0001; MADB106 - F(4,47)=9.479, p<0.0001). (Fig. 4B/C).

Changes in blood and lymph nodes leukocyte numbers, activation markers, and cytokine secretion following GLA-SE administration

Procedure

Seventy-three 4-months old F344 rats (34 females and 39 males) received either PBS or GLA-SE (2μg/animal) (s.c. 100μl) 1h, 4h, 12h, or 24h before sample collection. At time 0h, peripheral blood and inguinal lymph nodes were collected for FACS and ELISA analyses.

Results:

Circulation

GLA-SE elevated total lymphocytes and T cell numbers at 4h, which returned to baseline levels thereafter (12h and 24h) (p<0.05). Granulocyte numbers increased at 4h, more so at 12h (p<0.0001), and returned to baseline at 24h. (ANOVA: Lymphocytes - F(4,67)=3.634, p<0.01; T cells - F(4,67)=3.734, p<0.01; Granulocytes - F(4,67)=55.179, p<0.0001) (Fig. 5A/B/E). Numbers of granulocytes expressing NKG2D or FasL increased at 4h (p<0.05), and returned to baseline at 12h (ANOVA: NKG2D - F(4,67)=10.345, p<0.0001; FasL - F(4,67)=5.143, p<0.01). No changes were evident in circulating monocytes and NK# (Fig. 5C/D). IL-6 levels increased 24h following GLA-SE administration (p<0.05) (ANOVA: F(4,65)=3.094, p<0.05) (Sup. Fig. 7C).

Figure 5
The effects of GLA-SE (2μg/animal) on various immunocytes sub-sets in the circulation and the lymphatic system (sampled from the inguinal lymph nodes). In the circulation, increased numbers, when evident, occurred at the 4h time point, returning ...

Lymph nodes – A time dependent elevation in total lymphocytes, T cells, monocytes, and NK cell numbers was evident beginning at 12h following GLA-SE administration (p<0.05 to all) (ANOVA: lymphocytes - F(4,60)=4.923, p<0.01; T cells - F(4,60=3.447, p<0.05; monocytes - F(4,60)=4.677, p<0.01; NK - (F(4,60)=4.690, p<0.01) (Fig. 5F/G/H/I). IL-12 levels decreased at 4h, 12h, and 24h following GLA-SE administration (p<0.05) (ANOVA: F(4,59)=3.296, p<0.05), IL-6 baseline levels were up to 3-fold higher than in the circulation, and increased 24h following GLA-SE administration (p<0.01) (ANOVA: F(4,61)=3.248, p<0.05), and no changes in the pro-inflammatory cytokine IL-1β were evident (Sup. Fig. 7D/E/F).

A single GLA-SE injection efficiently reduced the in vivo formation of experimental metastases in rats and mice

To test the biological and clinical relevance of our findings, we assessed the in vivo long term effects of a single intermediate dose of GLA-SE on the actual development of experimental cancer metastases.

Procedure

In experiment 1, eighty-six 6-month old F344 rats (44 females and 42 males) received either PBS, SE, or GLA-SE (2μg/animal) (s.c. 100μl). 24h later, rats were injected i.v. with MADB106 tumor cells, and 3 weeks later sacrificed for the assessment of pulmonary MADB106 metastases (Materials & Methods).

In experiment 2, thirty-six 8-week old male BALB/c mice received PBS or GLA-SE (5μg/animal) (s.c. 100μl), either 24h before, or 24h after they were injected with CT26 tumor cells. Hepatic CT26 metastases were enumerated 3 weeks later (Materials & Methods).

Results

In experiment 1, the GLA-SE caused 40% reduction in the overall number of metastases compared to PBS or SE (p<0.05) (ANOVA: F(2,83)=5.405 , p<0.01) (Fig. 6A).

Figure 6
A single 2μg/animal GLA-SE injection in rats and a 5μg/animal GLA-SE injection in mice a day prior to tumor inoculation, reduced the number of (A) MADB106 lung metastases, and (B) CT26 hepatic metastases. Administration of GLA-SE a day ...

In experiment 2, GLA-SE administered 24h prior to tumor inoculation caused 75% reduction in number of liver metastases (ANOVA: F(2,33)=3.984, p=0.028), while GLA-SE administered 24h following tumor inoculation had no significant (p=0.092) effect on hepatic metastases (Fig. 6B).

Discussion

In the current study we found that GLA-SE rapidly elevates both innate and adaptive immunity in the circulation, lungs, and the lymphatic system. Additionally, GLA-SE reduced metastatic development of a mammary adenocarcinoma and a colon carcinoma by approximately 40-75% in F344 rats and BALB/c mice, respectively, but only when administered prior to surgery. Increased NK cell activity and numbers by GLA-SE was crucial in mediating at least some of these anti-metastatic effects, as selective depletion of NK cells in rats completely abolished GLA-SE's beneficial effects in this model. Furthermore, our results indicate that the doses of GLA-SE employed herein had no adverse effects, suggesting potential clinical use of this immunostimulatory agent in the perioperative context, when usually immune stimulation is contraindicated.

Specifically, a single 2μg subcutaneous injection of GLA-SE was sufficient for increasing the numbers and/or efficacy of NK, granulocytes, monocytes, and T cells in the circulation, the marginating-pulmonary compartment, and the lymphatic system. With the exception of the 4h time point, in which reduced NK cell numbers (but not cytotoxicity per cell) was evident in the circulation, all other time points and immune indices showed increased levels as early as 4 hours following inoculation, and some have been maintained for 24-48h. Most importantly, a single injection of GLA-SE increased host resistance to metastasis at all examined time intervals (including the 4h), markedly so between 12 to 48h post administration. Moreover, a single GLA-SE treatment 24h prior to MADB106 or CT26 tumor inoculations had long lasting effects, decreasing the actual number of lung/hepatic metastases three weeks following tumor inoculation. On the other hand, a single administration of GLA-SE 24h following tumor inoculation did not significantly improve CT26 outcomes, suggesting that either GLA-SE may be more effective against circulating tumor cells than established metastases, or that surgical stress responses may have dampen the efficacy of immune stimulation36.

Considering the host and its various immune compartments as a whole, the aforementioned alterations in immune competence induced by GLA-SE suggest specific trafficking patterns that aim at enhancing immunological capabilities in strategic organs. Specifically, a reduction in NK cell numbers in the circulation was evident during the first 12h following GLA-SE administration, but occurred concurrently with an elevation in their numbers and cytotoxicity in the lungs and the lymph nodes beginning at 12h following treatment. T cell numbers were elevated in the circulation only at the 4h time point following GLA-SE administration, followed by elevation in their numbers in the lymph nodes beginning at 12h and through 24h following treatment. These findings suggest that GLA-SE rapidly generates an immune reaction, resulting in NK and T cells migration from various organs through the circulation, towards susceptible and/or specialized organs, such as the lungs and the lymphatic system, where the host immune system commonly interacts with infection agents and circulating malignant cells. We hypothesize that these processes enable an effective systemic immunological response. Indeed, when assessing systemic resistance to metastasis, GLA-SE (2.0μg /animal) was beneficial at any time point tested (0h, 4h, 12h, 24h, and 48h). These findings are in contrast to our previous results, where employing LPS in this same model system rapidly induced deleterious effects following its administration, even when used at very low doses (0.3μg LPS/animal)20.

As GLA was developed to minimize Th2 side effects while maintaining an efficient Th1 reaction21, we tested potential adverse effects of GLA-SE in the doses found to be effective herein. No weight loss was observed following GLA-SE administration of either the 2μg dose used in most studies, or a higher dose of 10μg, as opposed to a marked weight reduction in animals receiving a standard effective dose of the TLR-3 agonist Poly I:C. Additionally, no behavioral changes in normal social exploratory activity were evident in animals receiving GLA-SE, in contrast to a marked effect of Poly I:C. Last, no elevation in the pro-inflammatory cytokine IL-1β was evident following GLA-SE administration. These findings have clinical ramifications, as mild and severe adverse effects, capable of endangering patients, are common in clinical trials employing TLR agonists9,41,42. Supporting the potential perioperative use of GLA in cancer patients, no adverse effects of GLA have been reported in more than 1,000 subjects immunized thus far with GLA in ongoing phase I/II clinical trials.

The immediate impact of GLA-SE is another advantage for its use in cancer patients during the perioperative period. Immune stimulatory agents that are used for prolonged period were shown to enhance tumor proliferation43 and may select for more resistant residual disease following tumor excision12. Administrating GLA-SE a day prior to surgery is feasible and would induce the desirable immunostimulatory impact, but will not allow sufficient time for the potential deleterious effects vis-à-vis the malignant tissue induced by prolonged use of immunostimulatory agents.

Surgery for the excision of a primary tumor bears deleterious impact on the metastatic process in cancer patients12, and we hypothesize that GLA-SE could overcome some of them, and may improve long-term cancer outcomes. Specifically, whereas excision of a primary tumor is indispensable, growing evidence now suggest that the perioperative period, and surgery itself, constitute risk factors for metastatic progression11,12,44. The perioperative period is characterized by physiological and psychological stress responses, including elevated secretion of catecholamines, glucocorticoids, prostaglandins and various other substances45-47. Others and us have shown that these compounds often reduce immune efficacy, and consequently potentiate pro-metastatic characteristics of residual malignant cells and their surrounding tissues, rendering the host more susceptible to the metastatic process at the critical perioperative period48,49. For example, post-operatively, catecholamines and prostaglandins have been shown to suppress NK cells cytotoxicity in vivo33,36,47,50, and glucocorticoids have been shown to decrease circulating IL-12 levels, hence reducing Th1 differentiation45. GLA-SE may overcome some of these deleterious effects when administered prior to surgery, given its immune enhancing and metastatic reducing impact evident herein. As the TLR-4 receptor and its signaling cascade is highly conserved throughout evolution15, and given the above promising findings, we believe it will be important to further study the use of GLA-SE in various animal models of spontaneous metastases, and, in the clinical settings, as a potential immunotherapeutic agent during the perioperative period.

Supplementary Material

Supp FigureS1-S5

Supp MaterialS1

Acknowledgments

This work was supported by NIH/NCI grant # CA125456 (SBE).

Footnotes

Conflict of interest: Steve Reed is a shareholder in Immune Design Corp., which is granted a worldwide exclusive license for the research, development, and commercialization of IDRI's GLA technology for products targeting a number of indications.

Novelty & Impact Statements: The perioperative period presents an unexploited window of opportunity for interventions aiming at reducing the risk of cancer metastases – the major cause of cancer related death. However, immune stimulation has not been used perioperatively given several contraindications to surgery. GLA-SE is currently used in phase-I/II clinical trials for other indications, and our results indicate its potential use as an effective immune-stimulator, which can be employed perioperatively without adverse effects, leading to reduced cancer recurrence.

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