Exploiting a Type III Interferon Response to Improve Chemotherapeutic Safety and Efficacy

doi:10.21203/rs.3.rs-4289446/v1

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Exploiting a Type III Interferon Response to Improve Chemotherapeutic Safety and Efficacy

https://doi.org/10.21203/rs.3.rs-4289446/v1

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Immune reactions to nanomedicines can be detrimental to the patient and compromise efficacy. However, our recent study characterizing the effects of a type III interferon (IFN-λ) response to lipid nanoparticles complexed with nucleic acids (lipoplexes) suggests that an IFN-λ pretreatment can increase the efficacy of chemotherapeutic nanomedicines. In this study we sought to clarify which cell type(s) are capable of producing IFN-λ in response to lipoplexes and how the effects of IFN-λ are propagated. Additionally, we demonstrate that an IFN-λ pretreatment is also capable of altering the accumulation profile of small molecules like doxorubicin. Finally, we assessed different administration routes for an IFN-λ pretreatment and showed the ability of this pretreatment to significantly increase the survival time of mice receiving Doxil® in a murine CT26 tumor model. With several chemotherapeutic nanomedicines available in the clinic and an IFN-λ product recently completing late phase clinical trials, this study provides the model for a novel anti-cancer treatment regime that can be rapidly translated to the clinic and improve the efficacy of contemporary treatment protocols.

Lipoplex

Nanoparticle

Tumor Delivery

Type III Interferon

Cancer

Interferon Lambda

The immune system has represented one of the most formidable barriers to nanoparticle-mediated delivery for the past several decades. Immune reactions to nanomedicines such as the Accelerated Blood Clearance (ABC) phenomenon, hyperactivation and/or suppression, anaphylaxis, Mononuclear Phagocytic System (MPS) uptake, and a recently characterized systemic type III interferon (IFN-λ) response have all be reported^1–8. Many of these reactions can adversely affect the pharmacokinetics and efficacy of the nanomedicines. The most common solution that has been proposed is to employ a “stealth” coating and/or improve targeting to tumor tissues. Unfortunately, these approaches pose their own risks and can even exacerbate the previously mentioned reactions^5,6,8–10. However, the IFN-λ response is unique in that it does not seem to produce any negative toxicities but reduces accumulation of nanomedicines in healthy tissues while increasing accumulation in tumor tissues³.

Type III interferons (IFN-λ1: IL-29, IFN-λ2/3: IL-28A/B) are a family of antiviral cytokines responsible for regulating epithelial and endothelial cell barrier functions and supplementing type-I interferon responses^11–16. However, IFN-λ1 is a pseudogene in mice and only IFN-λ2/3 are expressed. The phenotypic changes in epithelial/endothelial cells brought on by IFN-λ leads to “tightening” of cellular junctions and limits the ability of viruses to penetrate and infect heathy tissues^17–22. In fact, a recently completed phase III clinical trial demonstrated that a prophylactic IFN-λ treatment can significantly reduce the infection and hospitalization rate due to COVID²³. Since type III interferons were only recently characterized, there has been little to no research done to investigate the interplay of IFN-λ and nanomedicine delivery. In this context, it is important to recognize a poorly understood “refractory response” to repeat administrations of nonviral gene therapy vectors complexed with lipid nanoparticles (lipoplexes). This response is characterized by an inability to increase either delivery and/or expression of the vector in the target tissues with repeated administrations of the lipoplex^24–32. This refractory state has been reported to initiate 24 h after the primary lipoplex injection and can last for up to 3 weeks^24–31. Due to the numerous physicochemical similarities between lipoplexes and viruses (e.g. nanosized, lipid membranes, nucleic acid components), we propose that the refractory response is an antiviral response to the lipid-nucleic acid complexes. As such, in our previous study we sought to determine if lipoplexes would elicit an antiviral IFN-λ reaction. Our results showed that an intravenous injection of lipoplexes results in high plasma levels of IFN-λ and significant alterations in the accumulation profile of a subsequently injected nanoparticle³.

When tumor-bearing mice are pretreated with either lipoplexes or exogenous IFN-λ 24 h before a dose of liposomal encapsulated doxorubicin (Doxil®), we observe a significant decrease in off-target accumulation of Doxil® with a concurrent increase in plasma concentration and tumor deposition when compared to mice that did not receive the pretreatment³. Since the primary off-target toxicity observed with Doxil® use is palmar-plantar erythrodysesthesia (blistering of the skin on the hands and feet)^33,34, it is of particular interest that accumulation of Doxil® in the skin of mice receiving a lipoplex/IFN-λ pretreatment was decreased dramatically³. This altered accumulation profile has the potential to not only reduce the most prominent off-target toxicity of Doxil® but increase the tumor delivery and efficacy of many different chemotherapeutic nanoparticles.

To further understand and characterize the effects of IFN-λ on nanoparticle biodistribution, we sought to compare epithelial and endothelial cells in terms of their ability to produce IFN-λ after lipoplex exposure and responsiveness to exogenous and/or endogenous IFN-λ. Additionally, we performed experiments to assess the effects of IFN-λ pretreatment on the biodistribution of a small molecule (doxorubicin). Finally, we confirmed the utility of two different routes of administration for an IFN-λ injection and investigated whether an IFN-λ pretreatment can increase the efficacy of Doxil®.

Animals:

Accumulation Experiments: Prior to treatment, female immunocompetent Balb/c mice 6–10 weeks old were acquired from Jackson labs (Bar Harbor, ME) and allowed to acclimate for one week. The mice were then inoculated in the right flank with approximately 1 million CT26 murine colon carcinoma cells (ATCC #CRL-2638). Tumors were allowed to grow for approximately 7 days or until tumor size reached at least 100 mm³. Mice were then randomized into treatment groups standardized to tumor volume. Each mouse received a single dose of 200 µL 1x PBS, 10 µg of recombinant mouse IFN-λ2 (R&D Systems, 4635-ML-025/CF) in 100 µL 1x PBS by a tail vein injection or subcutaneous (SubQ) injection in the dorsal region. Twenty-four hours after pretreatment, animals were administered a tail vein injection of 10 mg/kg liposomal doxorubicin (Doxil®; Sun Pharmaceutical Industries) or 10 mg/kg doxorubicin HCl acquired from Cayman Chemical (Ann Arbor, MI). Twenty-four hours after the Doxil®/doxorubicin injection, animals were euthanized, and organs collected for analysis.

Tumor Growth Inhibition Study: Mice were acquired and tumors inoculated as described above. Mice were then randomized into groups of five standardized to tumor volume. Each group received its treatment regime twice (1st treatment on days 0&1 after randomization, 2nd treatment on days 7&8). Treatment groups are as follows: 1) 100 µL 1x PBS SubQ followed 24 h later by a 200 µL 1x PBS tail vein injection 2) 100 µL 1x PBS SubQ followed 24 h later by a 10 mg/kg dose of Doxil® 3) 10 µg IFN-λ2 SubQ followed 24 h later by a 200 µL 1x PBS tail vein injection 4) 10 µg IFN-λ2 SubQ followed 24 h later by a 10 mg/kg dose of Doxil®.

Liposome/Lipoplex Preparation:

Sphingosine, cholesterol, 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DAPC), and C16-PEG750-Ceramide were purchased from Avanti Polar Lipids (Alabaster, AL) and used to prepare liposomes at a 3:2:4.5:0.5 mole ratio (respectively) or a 3:2:5 mole ratio for non-PEGylated liposomes as previously described³⁵. The liposome preparations have an average diameter of 135 nm, a polydispersity index (PDI) of 0.137, and were prepared in sterile ddH₂O. To form lipoplexes, liposomes were mixed with plasmid DNA (donated from Megabios Corp., Burlingame, CA) in sterile ddH₂O at a charge ratio of 0.5^35,36. The resulting lipoplexes have an average diameter of 176 nm and PDI of 0.084. A Malvern Zetasizer Nano-ZS was used for all size measurements.

Interferon Lambda Receptor 1 (IFNLR1) mRNA Expression Quantification by Reverse Transcription-Polymerase Chain Reaction (RT-qPCR):

Human Lung Microvascular Endothelial Cells (HLMVECs, 540-05A) purchased from Sigma-Aldrich® (Burlington, MA) were cultured in 6-well plates and left untreated or treated with 100 ng/mL recombinant human IL-29 (R&D Systems® 1598-IL-025) or 100 ng/mL IL-28A/B (R&D Systems® 8417-IL-025 / 5259-IL-025) for 24 h. Human Primary Liver Epithelial Cells (H-6044) purchased from Cell Biologics, Inc (Chicago, IL) were cultured in 6-well plates and left untreated or treated with 100 ng/mL recombinant human IL-29 (IFN-λ1), 100 ng/mL IL-28A/B (IFN-λ2/3), 1 µg/mL of lipoplexes, or media from cells treated with lipoplexes 24 h prior. Each treatment was done in triplicate (3 wells/treatment group) and lasted for 24 h. After 24 h, using TaqMan™ Fast Advanced Cells-to-CT™ kit purchased from Invitrogen™ (Waltham, MA), cells were collected according to manufacturer recommendations and IFNLR1 expression was quantified using the manufactures recommended protocol and instruments. PCR primer Hs00417120_m1 was used to quantify IFNLR1 gene expression and PCR primer Hs03928985_g1 was used to quantify reference gene RNA18S5 expression. Primers were purchased from Invitrogen™.

IL28A/B Quantification by ELISA:

Human Primary Liver Epithelial Cells were cultured and left untreated or treated with 0.25 µg/mL lipoplexes, 0.5 µg/mL lipoplexes, or 1 µg/mL lipoplexes for 24 h. IL28A/B (IFN-λ2/3) levels were then determined through enzyme-linked immunosorbent assay (ELISA) using the manufacturer’s recommendations and protocols. Mouse IL-28A/B (IFN-lambda 2/3) DuoSet® ELISA (DY1789B-05) was purchased from R&D Systems® (Minneapolis, MN). ELISA Lower Limit of Quantification (LLoQ) = 31.30 pg/mL. ELISA Lower Limit of Detection (LLoD) = 3.90 pg/mL.

Quantification of Doxorubicin Fluorescence and Organ Accumulation:

A standard curve of fluorescence for doxorubicin was determined through fluorescence measurements of a serially diluted stock solution of doxorubicin in 90% isopropyl alcohol (IPA) acidified with HCl to a final concentration of 0.075 M. Fluorescence measurements were conducted in a CellStar™ µClear™ 96-Well, Cell Culture-Treated, Flat-Bottom Microplate from Greiner Bio-One™ (Monroe, NC) at an excitation of 470 nm and emission of 595 nm using a Molecular Devices (San Jose, CA) SpectraMax M5. To determine doxorubicin levels in whole organs and tissues (lung, heart, liver, spleen, kidneys, brain, tumor, skin, plasma), blank tissues were run through an extraction procedure using 90% IPA/0.075 M HCl as described below. Once extracted, the blank organ extract was used as a buffer to serially dilute a stock solution of doxorubicin. The equation from the resulting standard curve of fluorescence from each specific organ was used to calculate the total amount of doxorubicin extracted from a corresponding Doxil®/doxorubicin-treated organ.

Extraction of Doxorubicin from tissues:

Organs collected from animals were immediately flash-frozen in liquid nitrogen and stored at -80 ⁰C until analysis. Organs were thawed, weighed, and extracted using a buffer made of 90% IPA/0.075 M HCl. Briefly, each organ was placed in a 2-ml tube containing lysis matrix A (MP Biomedical, 6910500) (each liver was equally divided into four tubes) and 1 mL of 90% IPA/0.075 M HCl was then added to each tube. Organs were then homogenized using a MiniBeadBeater-16 Model 607 from BioSpec Products (Bartlesville, OK). Tubes were shaken for 90 sec then allowed to rest on ice for 5 min before another 90 sec shake. Once homogenized, the tubes were allowed to rest on ice for 5 min before being centrifuged at ~ 15,000 RCF for 15 min at 4°C. The supernatant was aspirated into a new tube and total volumes for all organs, except livers, were standardized to 1 mL using 90% IPA/0.075 M HCl. Divided liver supernatants were added together and standardized to 4 mL using 90% IPA/0.075 M HCl. Doxorubicin fluorescence of the extracted solution was then measured, and total doxorubicin accumulation was then calculated using the doxorubicin fluorescence standard curves described above. Based off our own extraction experiments and previous literature, an extraction efficiency of > 95% was assumed for all extraction calculation³⁷.

Statistics/Data availability:

For all accumulation comparisons, an unpaired t-test with Welch’s correction was performed. For tumor growth inhibition and weight loss comparisons a 2-way ANOVA with Mixed-Effects was performed. For survival comparisons, a Log-Rank (Mantle-Cox) test was performed. All statistical models were performed using GraphPad Prism version 9.5.0 for Windows, GraphPad Software, San Diego, California USA, www.graphpad.com. The data generated in this study are available upon request from the corresponding author.

Endothelial/Epithelial Cell Interferon Lambda Receptor 1 (IFNLR1) Expression

To test whether microvascular endothelial cells and/or liver epithelial cells can respond to IFN-λ, real-time quantitative polymerase chain reaction (RT-qPCR) analysis was used to determine IFNLR1 gene expression in primary Human Lung Microvascular Endothelial Cells and Human Primary Liver Epithelial Cells.

No expression of IFNLR1 was detected in basal state endothelial cells (Table 1). There was also no expression detected in endothelial cells treated with IFN-λ1 or IFN-λ2&3 (Table 1). In contrast, human liver epithelial cells show expression of IFNLR1 at basal state and show an increase in gene expression of 2.1-fold when treated with IFN-λ1 and an increase of 3.8-fold when treated with IFN-λ2&3 (Table 2). Liver epithelial cell IFNLR1 expression was also increased by 5-fold when treated with lipoplexes (Table 2).

Table 1

qPCR of IFNLR1 gene expression in primary human lung microvascular endothelial cells after IFN-λ treatment
Treatment	ΔCt mean	ΔCt SD	ΔCt SE	ΔΔCt	2^-ΔΔCt (Relative fold-change compared to untreated)
IFN-λ1 (IL29)	No Amplification Detected	n/a	n/a	n/a	n/a
IFN-λ2/3 (IL28A/B)	No Amplification Detected	n/a	n/a	n/a	n/a
Untreated	No Amplification Detected	n/a	n/a	n/a	n/a

Table 2

qPCR of IFNLR1 gene expression in primary human liver epithelial cells after lipoplex/IFN-λ treatment
Treatment	ΔCt mean	ΔCt SD	ΔCt SE	ΔΔCt	2^-ΔΔCt (Relative fold-change compared to untreated)
IFN-λ1 (IL29)	19.476	0.313	0.128	-1.092	2.132
IFN-λ2/3 (IL28A/B)	18.641	0.256	0.105	-1.972	3.803
Lipoplex	18.239	0.355	0.145	-2.330	5.027
Untreated	20.568	0.438	0.179	0	1.000

Quantification of Liver Epithelial Cell IFN-λ Production in Response to Lipoplex Treatment

Considering the responsiveness of liver epithelial cells shown in Table 2, we tested whether these cells are also capable of producing IFN-λ in response to lipoplex treatment. Accordingly, human primary liver epithelial cells were treated with varying amounts of lipoplexes for 24 h and cell media was then collected for determination of IFN-λ levels by ELISA. Media from liver epithelial cells treated with 1, 0.5, or 0.25 µg/mL lipoplexes showed an average IFN-λ level of 49.57, 33.52, and 22.42 pg/mL 24 h after treatment, respectively (Fig. 1). However, this latter value is below the lowest concentration that can be reliably quantified by the ELISA (LLoQ = 31.30 pg/mL).

Paracrine Signaling Potential of Liver Epithelial Cell Produced IFN-λ

Table 3: qPCR of INFLR1 gene expression in primary human liver epithelial cells after treatment with conditioned media

Treatment

ΔCt mean

ΔCt SD

ΔCt SE

ΔΔCt

2^-ΔΔCt

(Relative fold-change compared to untreated)

Media from cells treated with lipoplexes

16.526

0.501

0.290

-1.001

2.001

Untreated

17.527

0.076

0.044

1.000

Effect of Different IFN-λ Administration Routes on Doxil® Accumulation

To evaluate the effect of different administration routes for the IFN-λ pretreatment, mice were given 10 µg IFN-λ via intravenous or subcutaneous injection 24 h prior to Doxil® treatment. Twenty-four hours following Doxil® administration, tissues were harvested from experimental mice and the total Doxil® accumulation in tissues was determined using the extraction method and standard curves described above. Our results demonstrate that a subcutaneous injection of IFN-λ produces comparable effects to an intravenous injection in the heart, liver, spleen, and tumor (Figs. 2 & 3). The skin, kidney, lung, and plasma of the mice receiving a subcutaneous pretreatment all showed greater effects than the intravenous pretreatment group (Figs. 2 & 3). A trend toward increased tumor accumulation was also observed but was not statistically significant under our experimental conditions. PBS and IV 10 µg IFN-λ pretreatment data were previously published³ and are presented here to compare with SubQ pretreatment data.

Effect of IFN-λ Pretreatment on the Accumulation of Doxil® 72 Hours After Injection

To determine IFN-λ’s effect on the accumulation profile of Doxil® 72 h after injection, mice were given 10 µg IFN-λ subcutaneously 24 h prior to Doxil® treatment. Seventy-two hours following Doxil® administration, tissues were harvested from experimental mice and the total Doxil® accumulation in tissues was determined using the

extraction method and standard curves described above. Our results demonstrate that an IFN-λ pretreatment, when compared to PBS pretreatment, had no significant effect on the accumulation of Doxil® in the lung, liver, kidney, heart, plasma, and tumor 72 h after injection (Fig. 4). However, Doxil® accumulation in the spleen of mice pretreated with IFN-λ was significantly lower than mice receiving PBS pretreatment. There was no Doxil® detected in the skin at 72 h. Doxil levels in plasma and tumor exhibited the same trends as observed after 24 h (Fig. 3), but these differences were not statistically significant.

Effect of IFN-λ Pretreatment on the Accumulation of Doxorubicin HCl in Organs and Tumors

Considering the effects on Doxil® accumulation described above, we performed experiments to assess the effect of IFN-λ pretreatment on the distribution of free doxorubicin. In these experiments, mice were given a dose of IFN-λ followed 24 h later by a 10 mg/kg dose of free doxorubicin-HCl. Twenty-four hours following doxorubicin administration, tissues were harvested from experimental mice and the total doxorubicin accumulation in tissues was determined using the extraction method and standard curves described above. Our results demonstrate that a SubQ 10 µg IFN-λ pretreatment significantly reduces doxorubicin accumulation in the spleen, lung, kidney, and liver when compared to mice pretreated with PBS (Fig. 5). The heart also showed a trend toward decreased accumulation while the tumor tissues showed a trend toward increased accumulation. However, the heart and tumor differences were not statistically significant under our experimental conditions. There was no free doxorubicin detected in the plasma or skin at 24 h.

Effect of IFN-λ Pretreatment on the Efficacy of Doxil®

To evaluate whether an IFN-λ pretreatment can decrease toxicity and/or increase the efficacy of Doxil®, randomized tumor-bearing mice were given a dose of IFN-λ or PBS followed 24 h later by a 10 mg/kg dose of Doxil® or PBS on the day of randomization and the following day, respectively. Mice were given the same treatment regime one week later. Our results show a nonsignificant (p = 0.1372) difference in tumor growth inhibition when mice were treated with both IFN-λ and Doxil® when compared to mice only treated with Doxil® (Fig. 6). However, mice receiving the IFN-λ pretreatment exhibited significantly lower Doxil® toxicity (as indicated by weight loss). Strikingly, mice receiving the IFN-λ pretreatment had a significantly higher probability of survival and prolonged survival time (Chi square: 16.45, df: 3, p = 0.0009).

Immunological reactions to nanoparticles represent a major barrier to efficient nanoparticle-mediated drug and gene delivery. Most of these reactions result in undesirable inflammatory responses, increased uptake by circulating and tissue-resident immune cells, as well as decreased circulation times and delivery of the therapeutic particle^{1,2,4–7,9,10,31,35,36}. However, with nanoparticle-mediated vaccines rising to prominence due to the COVID-19 pandemic, utilizing nanoparticles to initiate beneficial immune responses is now a major area of focus for the nanoparticle community. One such immune response that has been mostly overlooked is an innate immune reaction to complexed gene therapy vectors (i.e., lipoplexes). This immune response was first described as a “refractory response” to repeatedly administered lipoplexes and resulted in the inability to achieve therapeutic levels of expression in target tissues^24–31. Our recently published work demonstrates that this refractory response is a systemic antiviral IFN-λ response that it is dependent on the presence of nucleic acids in the nanoparticle formulation³. Additionally, we demonstrated that both a lipoplex and IFN-λ pretreatment can significantly reduce the accumulation of 150 kDa dextran and/or Doxil® in healthy tissues³.

To understand how IFN-λ is capable of altering particle biodistribution, we refer to a study published by Lazear et al. in 2015 demonstrating that IFN-λ is capable of significantly increasing blood-brain-barrier (BBB) resistance and tight junction protein colocalization while decreasing permeability¹⁹. The enhanced barrier capabilities that are brought on by IFN-λ play a key role in limiting pathogen invasion of vital tissues like the nervous system and respiratory/gastrointestinal tracts^17–22. Due to these observations, we hypothesized that endothelial cells are the primary responders to IFN-λ, tightening their cellular junctions to restrict large particles from entering the underlying tissues. To test this hypothesis we probed the expression levels of IFN-λ Receptor (IFNLR1) in Human Lung Microvascular Endothelial Cells. Lung endothelial cells were chosen due to the respiratory tract being the primary entry site for many different viruses and as such represents an excellent target for the barrier enhancing effects of IFN-λ. Interestingly, we did not observe any expression of IFNLR1 in either basal state cells or cells treated with exogenous IFN-λ (Table 1). However, gene expression profiles of cultured endothelial cells are notoriously variable depending on environmental factors such as physical forces and heterotypic cell interactions^38,39. Whether human lung microvascular endothelial cells express the IFNLR1 has not been extensively investigated, but this variability in cultured endothelial cells may explain why we do not see IFNLR1 expression in our cell culture model.

Since lung endothelial cells, in our hands, did not show IFNLR1 expression, we turned our attention to epithelial cells. This is supported by the fact that IFN-λ is primarily produced by epithelial cells and directly regulates mucosal barrier epithelial cell permeability^21,40,41. Since the liver is the primary organ of the Mononuclear Phagocytic System (MPS) coupled with the critical role of IFN-λ in the hepatic immune response to hepatotropic viruses^42–46, we hypothesized that liver epithelial cells would play a key role in the altered biodistribution of nanoparticles observed after a lipoplex or IFN-λ pretreatment. To test this hypothesis we probed the expression of IFNLR1 in Human Primary Liver Epithelial Cells known to be responsive to IFN-λ⁴⁷. Our results show IFNLR1 expression in basal state liver cells with a ~ 2–3-fold increase in expression when treated with exogenous IFN-λ and a ~ 5-fold increase in expression when treated with lipoplexes (Table 2). We also provided evidence of the ability of endogenously produced IFN-λ to increase the expression of IFNLR1 by ~ 2-fold in naive liver epithelial cells (Table 3). This illustrates the ability of endogenously produced IFN-λ to propagate its effects through paracrine signaling. Collectively, these data suggest that human liver epithelial cells play a critical role in initiating and propagating an antiviral IFN-λ response. Interestingly, it has been repeatedly confirmed that mouse livers, unlike human livers, only weakly respond to and express IFN-λ and its receptor^48–51. Yet, we clearly demonstrate a significant effect of IFN-λ pretreatment on hepatic accumulation of both Doxil® and free doxorubicin in our Balb/c mouse model (Figs. 2 & 5). This suggests that IFN-λ in mice may exert its effects through different functions in different tissues. It may also be possible that downstream products of the IFN-λ response are the driving force behind the altered accumulation profiles. Future studies will need to more fully explore and elucidate the molecular mechanisms of IFN-λ.

Having to receive an intravenous injection of lipoplexes or IFN-λ as a pretreatment the day before another intravenous injection of a chemotherapeutic would present an uncomfortable and logistical burden to both the patient and healthcare providers. Accordingly, we considered if a subcutaneous (SubQ) injection of IFN-λ might be suitable as an effective pretreatment. Notably, a SubQ injection of IFN-λ was used as a prophylactic treatment for COVID-19 in a recently completed phase III clinical trial²³. To determine if SubQ administration of IFN-λ produces the same magnitude of effects we see with an intravenous injection, Doxil® organ/tissue accumulation in mice receiving a SubQ IFN-λ pretreatment was compared to Doxil® accumulation in mice receiving either PBS or an equivalent intravenous dose of IFN-λ as a pretreatment. Mice receiving SubQ IFN-λ showed comparable or greater effects when compared to mice receiving intravenous IFN-λ (Figs. 2 & 3). These data indicate that a SubQ injection would be the superior route for an IFN-λ pretreatment. In this scenario, a prefilled syringe of IFN-λ could be dispensed by a local pharmacy and patients can self-administer SubQ injections in the comfort of their own home and reduce the overall cost/time of treatment. Since it has been reported that IFN-λ induces rapid phenotypic changes in tissues and cells^19,52,53, it may not be necessary to wait 24 h between an IFN-λ pretreatment and subsequent chemotherapy treatment. If it takes only a few minutes or hours for IFN-λ to fully exert its effects, then a pretreatment could be administered the same day as the chemotherapeutic, further reducing patient burden. We plan to characterize the timing of the IFN-λ-induced tightening in future studies.

To determine how long the altered accumulation profile of Doxil® persists after an IFN-λ pretreatment, the organ/tumor accumulation of Doxil® was measured 72 h after a SubQ IFN-λ + Doxil® treatment regime. Interestingly, only the spleens of IFN-λ treated mice showed significantly lower accumulation of Doxil® when compared to PBS treated mice. All other major organs showed no significant difference between treatment groups after 72 h. Tumor tissues of IFN-λ treated animals showed a trend toward increased accumulation but this difference was not significant. These data suggest that IFN-λ’s effects on nanoparticle biodistribution may be short lived. It is possible that repeat administrations of IFN-λ may enhance or prolong the tightening response. As such, future studies should aim to determine the time needed for an IFN-λ pretreatment to elicit the tightened phenotype and how long this phenotype lasts. It is important to note that the clinical trials conducted with IFN-λ have all utilized the PEGylated version of the protein^23,54, which is not commercially available in either the human or murine form. It is expected that PEGylation would retard clearance rates, prolonging the tightened phenotype.

Due to the clear effects of IFN-λ on the biodistribution of nanosized particles, we sought to investigate whether IFN-λ can alter small molecule distribution. Surprisingly, mice receiving the IFN-λ pretreatment prior to a dose of doxorubicin-HCl exhibited reduced doxorubicin accumulation in major organs when compared to mice receiving no pretreatment (Fig. 5). It seems unlikely that IFN-λ is capable of tightening epithelial junctions so completely that not even small molecules can penetrate. Considering that approximately 75% of doxorubicin is bound to plasma protein⁵⁵, an alternative explanation would be that this altered biodistribution is due to reduced accumulation of protein-bound doxorubicin. Because many drugs circulate as protein-bound complexes, this phenomenon may be applicable to other small molecule drugs. However, more testing would be necessary to establish the effect of IFN-λ pretreatment on small molecule biodistribution.

The altered biodistribution of Doxil® following an IFN-λ pretreatment suggests that this treatment regime could lead to greater efficacy and reduced toxicity of Doxil®. To test this hypothesis we compared the tumor growth inhibition, weight loss, and survival times of mice receiving Doxil® with or without IFN-λ pretreatment. Mice receiving the IFN-λ pretreatment showed a slightly greater inhibition of tumor growth, minimal weight loss, and a significant increase in survival when compared to mice that did not receive the pretreatment (Fig. 6). All the mice that did not receive the IFN-λ pretreatment had to be euthanized due to either tumor ulcerations (n = 2) or weight loss greater than 15% (n = 3). Of the mice receiving the IFN-λ pretreatment, one mouse was found dead in its cage due to unknown causes, one mouse was euthanized due to tumor ulcerations, one mouse was removed from the study due to failure to properly administer the second treatment regime, and the other two mice survived until their tumors began to grow in volume again, at which point the mice were euthanized. The slightly greater inhibition in tumor growth that was observed could possibly be explained by the trend toward increased tumor accumulation of Doxil® we see in mice pretreated with IFN-λ. Likewise, the reduced off-target accumulation of Doxil® observed in mice receiving the IFN-λ pretreatment might explain the minimal weight loss and increase in survival times under these conditions. The data presented here clearly demonstrate the capability of an IFN-λ pretreatment to significantly reduce the toxicity and adverse outcomes of Doxil® while increasing its efficacy.

There are several limitations to our study that must be considered when interpreting our results and planning subsequent studies. For our in vitro cell models we only used primary human cells which are prone to variable gene expression profiles. To confirm our studies and further investigate the role of endothelial/epithelial cells in the IFN-λ response, a variety of cell lines under variable conditions would need to be probed for IFNLR1 expression and IFN-λ production. Regarding our tumor growth inhibition model, only female Balb/c mice were used and only a single CT26 solid tumor model was investigated. Future studies will need to address the effects of sex, tumor model, tumor metastasis, and strain/species differences. A recently developed IFNLR1 deficient mouse model⁵⁶ would be an excellent tool to help evaluate how IFN-λ influences nanomedicine delivery. Additionally, it would be beneficial to investigate how a systemic antiviral response to a pathogen like the coronavirus might affect drug distribution and pharmacokinetics. We hope our work begins to bring recognition to the potential anti-cancer utility of type III interferons and their profound effects on drug delivery.

In conclusion, our results demonstrate that human liver epithelial cells can produce IFN-λ in response to lipoplex treatment and upregulate IFNLR1 in response to both exogenous and endogenous IFN-λ. Furthermore, media from lipoplex-treated cells elicited IFNLR1 expression in naïve cells, supporting the notion that paracrine signaling plays a role in propagating the tightening phenotype. Additionally, we demonstrate the potential of a SubQ IFN-λ pretreatment to significantly increase the safety and efficacy of Doxil®, and presumably other chemotherapeutics. Finally, we provide evidence that an IFN-λ pretreatment can also significantly alter the biodistribution of the small molecule drug doxorubicin. Future studies will aim to further characterize and understand how to harness the unique properties of IFN-λ.

Ethics approval: All animal procedures were approved by the University of Colorado Institute for Animal Care and Use Committee in accordance with guidelines from the National Institutes of Health (USA).

Consent to participate and consent for publication: This was not a human subject study and therefore no consent to participate or for publication was necessary.

Competing interests: The authors have no relevant financial or non-financial interests to disclose.

Funding: This work was supported by the National Institutes of Health grants R01 GM129046 and K22 AI146141.

Authors’ Contributions: All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Scott Tilden. The first draft of the manuscript was written by Scott Tilden and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Acknowledgements: We are very grateful to Dr. Dmitri Simberg for supplying Doxil® and some of the mice used in our experiments.

Data Availability Statement: The data generated in this study are available upon request from the corresponding author.

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Exploiting a Type III Interferon Response to Improve Chemotherapeutic Safety and Efficacy

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Abstract

Figures

Introduction

Materials and Methods

Animals:

Liposome/Lipoplex Preparation:

Interferon Lambda Receptor 1 (IFNLR1) mRNA Expression Quantification by Reverse Transcription-Polymerase Chain Reaction (RT-qPCR):

IL28A/B Quantification by ELISA:

Quantification of Doxorubicin Fluorescence and Organ Accumulation:

Extraction of Doxorubicin from tissues:

Statistics/Data availability:

Results

Endothelial/Epithelial Cell Interferon Lambda Receptor 1 (IFNLR1) Expression

Quantification of Liver Epithelial Cell IFN-λ Production in Response to Lipoplex Treatment

Paracrine Signaling Potential of Liver Epithelial Cell Produced IFN-λ

Effect of Different IFN-λ Administration Routes on Doxil® Accumulation

Effect of IFN-λ Pretreatment on the Accumulation of Doxil® 72 Hours After Injection

Effect of IFN-λ Pretreatment on the Accumulation of Doxorubicin HCl in Organs and Tumors

Effect of IFN-λ Pretreatment on the Efficacy of Doxil®

Discussion

Conclusions

Declarations

References

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