Continuous sensing of IFNα by hepatic endothelial cells shapes a vascular antimetastatic barrier

Abstract
Editor's evaluation
eLife digest
Introduction
Results
Discussion
Materials and methods
Appendix 1
Data availability
References
Article and author information
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Abstract

Hepatic metastases are a poor prognostic factor of colorectal carcinoma (CRC) and new strategies to reduce the risk of liver CRC colonization are highly needed. Herein, we used mouse models of hepatic metastatization to demonstrate that the continuous infusion of therapeutic doses of interferon-alpha (IFNα) controls CRC invasion by acting on hepatic endothelial cells (HECs). Mechanistically, IFNα promoted the development of a vascular antimetastatic niche characterized by liver sinusoidal endothelial cells (LSECs) defenestration extracellular matrix and glycocalyx deposition, thus strengthening the liver vascular barrier impairing CRC trans-sinusoidal migration, without requiring a direct action on tumor cells, hepatic stellate cells, hepatocytes, or liver dendritic cells (DCs), Kupffer cells (KCs) and liver capsular macrophages (LCMs). Moreover, IFNα endowed LSECs with efficient cross-priming potential that, along with the early intravascular tumor burden reduction, supported the generation of antitumor CD8⁺ T cells and ultimately led to the establishment of a protective long-term memory T cell response. These findings provide a rationale for the use of continuous IFNα therapy in perioperative settings to reduce CRC metastatic spreading to the liver.

Editor's evaluation

This study describing how continuous perioperative IFNα therapy stimulates hepatic endothelial cells to build up a physical vascular barrier that limits tumor cell entry into the liver and promotes long-term antitumor immunity provides novel evidence for anti-metastatic effects of IFNα via effects on the liver vascular compartment. This work is predicted to advance the field and will be of interest to scientists studying cancer, inflammation and liver function.

https://doi.org/10.7554/eLife.80690.sa0

eLife digest

Colorectal cancer remains one of the most widespread and deadly cancers worldwide. Poor health outcomes are usually linked to diseased cells spreading from the intestine to create new tumors in the liver or other parts of the body. Treatment involves surgically removing the initial tumors in the bowel, but patient survival could be improved if, in parallel, their immune system was ‘boosted’ to destroy cancer cells before they can form other tumors.

Interferon alpha is a small protein which helps to coordinate how the immune system recognizes and deactivates foreign agents and cancerous cells. It has recently been trialed as a colorectal cancer treatment to prevent tumors from spreading to the liver, but only with limited success. This partly because interferon-alpha is usually administered in high and pulsed doses, which cause severe side effects through the body.

Instead, Tran, Ferreira, Alvarez-Moya et al. aimed to investigate whether continuously delivering lower amounts of the drug could be a better approach. This strategy was tested on mice in which colorectal cancer cells had been implanted into the wall of the large intestine. Continuous administration minimized the risk of the implanted cancer cells spreading to the liver while also creating fewer side effects. The team was able to identify an optimum delivery strategy by varying how much interferon-alpha the animals received and when.

Further experiments also revealed a new mechanism by which interferon-alpha prevented the spread of colorectal cancer. Upon receiving continuous doses of the drug, a group of liver cells started to generate a physical barrier which stopped cancer cells from being able to invade the organ. The treatment also promoted long-term immune responses that targeted diseased cells while being safe for healthy tissues. If confirmed in clinical trials, these results suggest that colorectal patients undergoing tumor removal surgery may benefit from also receiving interferon-alpha through continuous delivery.

Introduction

Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related death worldwide (Sung et al., 2021). Surgical resection of the primary CRC tumor is the mainstay of treatment (Argilés et al., 2020; Cunningham et al., 2010; Seo et al., 2013) unfortunately, up to 50% of these patients – despite chemotherapy and targeted adjuvant therapies – often develop life-threatening liver metastatic disease in the following years (Argilés et al., 2020; Sargent et al., 2009). While the overall benefit of surgery is well established (Seo et al., 2013), it has been also proposed that this procedure may foster liver metastases by increasing the dissemination of CRC cells into the portal circulation (Chow and Chok, 2019; Denève et al., 2013), enhancing the adhesion of CRC cells to the liver endothelium (Chambers et al., 2002; Gül et al., 2011) or promoting transient immunosuppression awakening dormant intrahepatic micrometastases (Ananth et al., 2016).

Accordingly, there is growing recognition that the use of perioperative immunotherapies in CRC patients undergoing surgical resection may represent a unique treatment window to prevent metastatic colonization and control minimal residual disease (Badia-Ramentol et al., 2021; Bakos et al., 2018; Horowitz et al., 2015). In this context, interferon-alpha (IFNα), a pleiotropic cytokine with multiple antitumor effects such as the direct inhibition of cancer cell growth and angiogenesis (Indraccolo, 2010), the sustained upregulation of major histocompatibility complexes (Gessani et al., 2014) and the induction of innate and adaptive antitumor immune responses (Aichele et al., 2006; Curtsinger et al., 2007; Fuertes et al., 2013), has been used as adjuvant immunotherapy in various solid cancers such as renal cell carcinoma (Flanigan et al., 2001), melanoma (Lens and Dawes, 2002) and colorectal cancer (Köhne et al., 1997; Link et al., 2005). Unfortunately, systemic administration of IFNα has shown limited clinical efficacy, likely due to its short plasma half-life (~1 hr) (Bocci, 1994) and the use of high and pulsed doses, which often resulted in systemic side effects (Weber et al., 2015). To overcome these limitations, several strategies to prolong IFNα half-life and target the tumor microenvironment have been tested (Fioravanti et al., 2011; Herndon et al., 2012; Jeon et al., 2013; Li et al., 2017; Liang et al., 2018; Yang et al., 2014), including a preclinical gene/cell therapy approach that can deliver constant amounts of IFNα into the liver to significantly curb CRC metastatic growth (Catarinella et al., 2016).

Herein, we adopted a continuous intraperitoneal (ip) IFNα delivery strategy to show that steady and tolerable IFNα doses reduce liver CRC metastatic spreading and improves survival in several CRC mouse models. Our results showed that the antimetastatic effects of IFNα rely neither on the direct inhibition of tumor cell proliferation nor on the indirect stimulation of hepatocytes, hepatic stellate cells, liver DCs, Kupffer cells (KCs) and liver capsular macrophages (LCMs). Rather, the results identify HECs, including LSECs, as key mediators of IFNα-dependent anti-tumor activities that involve the impairment of CRC trans-sinusoidal migration and the development of long-term anti-tumor CD8⁺ T cell immunity.

Results

Selection of the optimal IFNα dosing regimen

To avoid well-known toxicities, especially myelotoxicity, caused by high IFNα doses (Weber et al., 2015) and to define a delivery strategy providing prolonged and non-fluctuating IFNα levels in blood and tissues, normal inbred mice were implanted intraperitoneally with mini-osmotic pumps (MOP) constantly releasing different rates (i.e. 50 ng/day, 150 ng/day, or 1050 ng/day) of recombinant mouse IFNα1 (termed IFNα from now on) over time. Serum IFNα levels peaked at day 2 after MOP implantation and relative IFNα amounts (from ~100 pg/ml to ~1200 pg/ml) reflected the different MOP loading doses (Figure 1A). Serum IFNα levels decreased, albeit not uniformly, at days 5 and 7 post implantation (Figure 1A), mirroring the pharmacokinetic-pharmacodynamic (PK-PD) behavior of other long-lasting formulations of IFNα (Jeon et al., 2013). A reduction in circulating white blood cells (WBCs) but not in platelet (PLT) counts or hematocrit (HCT) was detected only with the highest dose (Figure 1B and Figure 1—figure supplement 1A,B). Looking at liver toxicity, we observed no increases in serum alanine aminotransferase (sALT) at all tested doses and time points (Figure 1—figure supplement 1C) and no abnormal changes in liver morphology at autopsy (Figure 1—figure supplement 1D). Looking at the intrahepatic induction of the interferon-stimulated gene (ISG) Irf7 (Cheon et al., 2014) at day 7, we observed a proportional dose response, with a six-fold increase in Irf7 expression at the 150 ng/day dosing regimen (Figure 1C). Notably, this increase paralleled the increase that we previously documented to be associated with protection against liver CRC colonization following a gene/cell therapy based IFNα delivery strategy (Catarinella et al., 2016). Lack of bone marrow and liver toxicity, proper induction of hepatic Irf7 expression and maintained responsiveness of hepatic liver cells to IFNα (Figure 1—figure supplement 1E) prompted us to select the 150 ng/day dosing regimen for follow-up investigations.

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Selection of the optimal IFNα dosing regimen.

(A) Quantification of plasma IFNα concentration from mice continuously treated with different IFNα doses at the indicated time points. Mean values ± SEM are shown; p-values were calculated by one-way ANOVA Tukey’s multiple comparison test. Significant p-values refer to the IFNα 50 ng/day group, since NaCl-treated animals had IFNα plasma levels below the assay detection limit. p≤0.05; **p≤0.01; ***p≤0.001. (B) White blood cell (WBC) counts of mice treated with different IFNα doses at indicated time points. Horizontal dashed lines delimit normal WBC range. Mean value ± SEM are shown; p-values were calculated by one-way ANOVA Tukey’s multiple comparison test. Significant p-values are referred to the NaCl group. *p≤0.05; **p≤0.01. (C) Quantitative real-time PCR analysis of *Irf7* mRNA expression from liver tissues of mice treated with different IFNα doses for 7 days. Basal *Irf7* mRNA expression level was determined in control mice (NaCl; n=9) and the relative expression of *Irf7* upon IFNα treatment is shown (n=3, for each IFNα-treated group). Mean values ± SEM are shown; p-values were calculated by Mann-Whitney test.

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Continuous IFNα administration reduces liver CRC metastatic burden and improves survival

We next tested the ability of continuous IFNα administration (150 ng/day for 28 days) to reduce CRC metastatic growth in the liver. Groups of H-2^bxd F1 hybrids of C57BL/6 J x BALB/c (CB6) mice were implanted with either control MOP-NaCl (termed NaCl) or MOP-IFNα (termed IFNα) (Figure 2A). Seven days later, a time frame compatible with the perioperative period in humans (Horowitz et al., 2015), CB6 mice were intrasplenically challenged with either the immunogenic microsatellite instable (MSI) MC38 CRC cell line (Efremova et al., 2018; Rosenberg et al., 1986) or the poorly immunogenic microsatellite stable (MSS) CT26 CRC cell line (Brattain et al., 1980; Efremova et al., 2018). Rapid removal of the spleen after CRC cell injection was implemented to avoid intrasplenic tumor growth (Catarinella et al., 2016). Each CRC cell line was injected at doses known to induce similar survival rates in age- and sex-matched CB6 recipients that, carrying hybrid H-2^bxd alleles, are immunologically permissive to both MC38 and CT26 cells (Catarinella et al., 2016). After treatment initiation, well-tolerated serum IFNα levels of ~300 pg/ml at day 2 and ~100 pg/ml thereafter were observed (Figure 2B and Figure 2—figure supplement 1A,B), which subsequently declined to undetectable levels. The intrahepatic expression of Irf7 monitored at day 21 after continuous IFNα therapy (Figure 2—figure supplement 1C, D) was like that observed earlier at day 7 (Figure 1C). Magnetic resonance imaging (MRI)-based longitudinal analyses – in MC38- (Figure 2C and Figure 2—figure supplement 1E) or CT26-challenged (Figure 2D and Figure 2—figure supplement 1F) animals – revealed that 100% of NaCl-treated mice (Figure 2E and F) develop multiple metastatic tumor lesions by days 21 and 28 after challenge and no mice displayed detectable tumors in other organs. Liver lesions increased in volume afterwards, ultimately resulting in imposed humane euthanization of both MC38 (Figure 2—figure supplement 1I) or CT26 (Figure 2—figure supplement 1J) tumor carriers in the intervening weeks. Conversely, 45% and 66% of IFNα-treated mice challenged with MC38 or CT26 cells, respectively, showed absence of liver metastases throughout the entire duration of the experiment (Figure 2E and F). All the remaining mice scoring disease-positive at days 21 and 28 displayed lesions that were reduced in number and size when compared to those detected in NaCl-treated counterparts (Figure 2—figure supplement 1G, J). Of note, metastatic lesions eventually regressed and achieved complete remission by day 50 in approximately 33% of IFNα-treated mice that were challenged with MC38 cells and scored disease-positive at day 21 (Figure 2C and E and Figure 2—figure supplement 1E), whereas none of the few CT26-challenged mice scoring disease-positive at day 21 survived long-term (Figure 2D and F and Figure 2—figure supplement 1F), with tumors confined only to the liver. Continuous IFNα administration also improved survival, with similar rates for both MC38- and CT26-challenged mice (Figure 2G and H). These results indicate that the continuous IFNα administration safely and efficiently limits the liver metastatic colonization of CRC cell lines intrinsically carrying different immunogenic or genetic properties.

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Continuous IFNα administration reduces liver CRC metastatic burden and improves survival.

(A) Schematic representation of the experimental procedure. Intrasplenic injection of 7x10⁴ MC38 or 5x10³ CT26 cells was performed 7 days after continuous NaCl or IFNα therapy. (B) Quantification of plasma IFNα concentration at different time points after continuous IFNα administration (n=6). The time of intrasplenic CRC cell injection has also been depicted. Mean values ± SEM are shown. (**C–D**) Representative T1 contrast-enhanced magnetic resonance images (MRI) of the liver of mice treated with NaCl (red frame) and IFNα (blue frame) at 21, 28, and 50 days after MC38 (C) or CT26 (D) cells injection. Red dashed lines highlight CRC liver metastases, characterized as hypointense regions in T1-weighted sequences. n.a.=not assessed, is referred to mice euthanized before the specified time point; scale bar = 5 mm. (**E–F**) Percentage of mice treated with NaCl (MC38 n=3 + 3; CT26 n=5 + 5 for each of two independent experiments) or IFNα (MC38 n=5 + 6; CT26 n=5 + 6 for each of two independent experiments) bearing at least one CRC liver metastasis estimated by MRI analysis at indicated time points after MC38 or CT26 injection. The oblique black line pattern within columns depicts the percentage of mice euthanized before the indicated time point. Mean values are shown; p-values were calculated by Fisher’s exact test. (**G–H**) Kaplan-Meier survival curves of Sham (n=3), NaCl- (MC38 n=6; CT26 n=10) or IFNα-treated (MC38 n=11; CT26 n=11) mice after MC38 or CT26 cells injection. The blue pattern indicates the time frame of IFNα ip release; p-values were calculated by log-rank/Mantel-Cox test.

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Continuous IFNα administration prevents spontaneous hepatic colonization of orthotopically implanted CT26^LM3 cells

To confirm the above-mentioned results in a different metastatic setting, we developed an orthotopic CRC model of liver metastases by implanting invasive CRC cells into the mouse cecal wall. As previously reported (Zhang et al., 2013), invasive CRC cells were generated by serial intracecal injections of the parental CT26 cells into CB6 mice (Figure 3—figure supplement 1A). The percentage of metastatic livers in intracecally implanted mice significantly increased as CT26 cells were passaged, with an almost 100% of animals bearing multiple liver metastases after 3 rounds of in vivo selection (Figure 3—figure supplement 1B-D). Three-time passaged cells (termed CT26^LM3) were then orthotopically implanted in the cecal wall of CB6 mice and 7 days later the animals were treated with either NaCl or IFNα (Figure 3A).

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Continuous IFNα administration prevents spontaneous hepatic colonization of orthotopically implanted CT26^LM3 cells.

(A) Schematic representation of the experimental procedure. Seven days after intracecal injection of 2x10⁵ CT26^LM3 cells, mice were randomly assigned to receive either continuous NaCl (n=7) or IFNα (n=6) administration and analyzed by MRI 14 days later. (B) Quantification of plasma IFNα concentration at the indicated time point after cecal wall injection of CT26^LM3 cells in mice described in a. The arrow indicates the time of NaCl or IFNα therapy initiation. Mean values ± SEM are shown. (C) WBC counts from mice described in (A) continuously treated with NaCl or IFNα at indicated time points. Horizontal dashed lines delimit the normal WBC range. The time of MOP implantation has also been depicted. Mean value ± SEM are shown; p-values were calculated by one-way ANOVA Tukey’s multiple comparison test. Significant p-values are referred to NaCl group. ***p≤0.001. (D) Representative images (top panels) of the hepatic lesions and intracecal tumors observed in NaCl- (red frame) and IFNα-treated (blue frame) mice, 21 days after CT26^LM3 cells intracecal wall injection, and the corresponding hepatic contrast-enhanced MRI, T1-weighted (middle panels) and T2-weighted (bottom panels) sequences. Red dashed lines identify macroscopic liver metastatic lesions. Scale bars = 5 mm. (E) Quantification of the weight of primary CRC tumors 21 days after CT26^LM3 cells intracecal wall injection of mice described in D. (F) Quantification of the number of hepatic lesions and total tumor volume of liver metastases by MRI analysis of mice described in D. Mean values ± SEM are shown; p-values were calculated by Mann-Whitney test. (G) Representative H&E, Ki67, CD34, F4/80, and CD3 immunohistochemical micrographs of liver metastatic lesions found in NaCl- (red frame) and IFNα-treated (blue frame) mice, 21 days after intracecal injection of CT26^LM3 cells. Scale bar = 100 μm. (H) Quantification of the percentage of mice bearing liver metastases, as well as the percentage of tumor area and the number of cells expressing Ki67, CD34, F4/80, and CD3 per mm² determined by IHC. Immunohistochemical measurements were conducted on at least 1000 mm² of total liver area for both experimental conditions. Mean values ± SEM are shown; p-values were calculated by Mann-Whitney test.

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Consistent with our previous results (Figure 2B), serum IFNα levels peaked at day 2 after MOP implantation (Figure 3B), without causing myelotoxicity (Figure 3C), and MRI analyses performed 14 days later revealed that continuous IFNα therapy did not alter the growth of primary intracecal tumors (Figure 3D and E), while IFNα treatment significantly reduced both number and size of hepatic lesions (Figure 3D and F) with 60% of mice spared from metastatic lesions (Figure 3H). The primary intracecal tumors (Figure 3—figure supplement 2A) and liver metastases (Figure 3G) detected after orthotopic implantation of CT26^LM3 cells were also characterized by immunohistochemistry (IHC). This analysis showed that primary intracecal tumors and liver metastatic lesions of NaCl-treated control mice were highly proliferative (as denoted by Ki67 positivity), exhibited marked signs of angiogenesis (as denoted by CD34 staining) and, accordingly with previous reports (Catarinella et al., 2016; Tauriello et al., 2018), were devoted of F4/80⁺ resident macrophages and CD3⁺ T cells (Figure 3G and H). Similar results were also observed in IFNα-treated primary intracecal tumors (Figure 3—figure supplement 2A, B). The absence of liver metastases in the majority of IFNα-treated mice is reflected by a reduced Ki67 or CD34 staining and an apparently normal distribution of F4/80⁺ macrophages and CD3⁺ T cells (Figure 3G and H). The few small hepatic lesions detected in 40% of mice continuously treated with IFNα (Figure 3H and Figure 3—figure supplement 2C, D) did not show differences in Ki67 positivity, CD34 staining or amount of F4/80⁺ resident macrophages and CD3⁺ T cells in relation to NaCl-treated mice (Figure 3—figure supplement 2C, D), consistent with the notion that CRC tumors may deregulate the Ifnar1 receptor and, thus, become refractory to IFNα therapy (Boukhaled et al., 2021; Katlinski et al., 2017).

Altogether, these results indicate that continuous IFNα therapy does not significantly alter the growth of primary established CRC tumors but reduces the liver metastatic potential of invasive CRC cells emerging from the cecum.

HECs mediate the anti-metastatic activity of IFNα

As the Ifnar1 surface receptor subunit is necessary to mediate the pleiotropic anti-tumor properties of IFNα (Cheon et al., 2014), we deleted this molecule from CRC cells and from hepatic parenchymal and non-parenchymal cells to identify the mechanism of action (MoA) of continuous IFNα administration in our in vivo system. We restricted these studies to MC38 CRC cells because the genetic background of the mouse models did not allow us to use CT26 CRC MHC-I mismatched cell lines in C57BL/6 recipients. We also adopted a new seeding approach that – involving the injection of CRC cells through the superior mesenteric vein – potentially avoids immune deregulations linked to the splenectomy procedure. Seven days after NaCl or IFNα administration, C57BL/6 mice were challenged with wild-type MC38 cells or with MC38 cells that were CRISPR-Cas9-edited to lack a functional Ifnar1 receptor (MC38^Ifnar1^_KO). To this end, MC38-edited clones showed mismatches in a T7E1 assay and clone C8 (MC38^C8) carrying Ifnar1 deleting mutations failed to express Irf7 upon in vitro IFNα stimulation (Figure 4—figure supplement 1A-C).

MRI analysis at day 21 after CRC challenge revealed that, in comparison with liver metastases observed in NaCl-treated controls, the lesions produced by MC38- or MC38^Ifnar1^_KO cells in IFNα-treated mice were similarly reduced in number and size (Figure 4A and C–D) and this resulted in comparable mouse survival rates (Figure 4E) in the absence of apparent myelotoxicity (Figure 4—figure supplement 2A). These data support the hypothesis that in our experimental setting the continuous IFNα administration has no direct antiproliferative activity towards CRC cells consistent with our previous reported data (Figure 4—figure supplement 1A-C; Catarinella et al., 2016).

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HECs mediate the antimetastatic activity of IFNα.

(A) Representative hepatic contrast-enhanced MRI of wild-type mice (C57BL/6) at day 21 after injection of 5x10⁴ MC38 cells or 5x10⁴ MC38^Ifnar1^_KO cells. Treatment with NaCl (red frame) and IFNα (blue frame) was initiated 7 days prior to intrasplenic injection of CRC cells. Red dashed lines highlight CRC liver metastases, characterized as hypointense and slightly-hyperintense regions in T1- and T2-weighted sequences, respectively. Scale bar = 5 mm. The number of mice for each experimental condition is also indicated. (B) Representative hepatic contrast-enhanced MRI at day 21 after MC38 intrasplenic injection of *Ifnar1^fl/fl* mice and mice lacking *Ifnar1* on hepatocytes (*Alb^Ifnar1*^_KO), hepatic stellate cells (*Pdgfrb^Ifnar1*^_KO), CD11c-expressing DCs/KCs/LMCs (*Itgax^Ifnar1*^_KO) and endothelial cells (*VeCad^Ifnar1*^_KO) injected with 5x10⁴ MC38 cells. Treatment with NaCl (red frame) and IFNα (blue frame) was initiated 7 days before MC38 cells intrasplenic injection. Red dashed lines highlight CRC liver metastases, characterized as hypointense and slightly-hyperintense regions in T1- and T2-weighted sequences, respectively. Scale bar = 5 mm. Number of mice for each experimental condition are also indicated. (C) Quantification of the number of hepatic lesions by MRI analysis, 21 days after CRC cells injection, of NaCl and IFNα-treated mice found in C57BL/6 mice, *Ifnar1^fl/fl* mice and all conditional *Ifnar1*_KO mouse models described in (A) and (B). Mean values ± SEM are shown; p-values were calculated by Mann-Whitney test. (D) Quantification of total tumor volume of liver metastases by MRI analysis at day 21 after CRC intrasplenic injection of NaCl- and IFNα-treated mice found in C57BL/6 mice, *Ifnar1^fl/fl* mice and all conditional *Ifnar1*_KO mouse models analyzed. Mean values ± SEM are shown; p-values were calculated by Mann-Whitney test. (E) Kaplan-Meier survival curves of C57BL/6 mice injected with MC38 cells (top) or MC38^Ifnar1_KO cells (bottom) described in (A). Sham injected animals (n=3) were used as control. The blue pattern indicates the time frame of IFNα ip release; p-values were calculated by log-rank/Mantel-Cox test. (F) Kaplan-Meier survival curves of the indicated groups of mice described in (B). Sham injected animals (n=3 per group) were used as control. The blue pattern indicates the time frame of IFNα ip release. The total number of mice for each experimental group were: NaCl-C57BL/6 n=8; IFNα-C57BL/6 n=6; NaCl-MC38^Ifnar1^_KO-C57BL/6 n=5; IFNα-MC38^Ifnar1^_KO-C57BL/6 n=6; NaCl-*Ifnar1^fl/fl* n=6; IFNα*-Ifnar1^fl/fl* n=11; NaCl-*Alb^Ifnar1*^_KO n=6; IFNα-*Alb^Ifnar1*^_KO n=8; NaCl-*Pdgfrb^Ifnar1*^_KO n=5; IFNα-*Pdgfrb^Ifnar1*^_KO n=7; NaCl-*Itgax^Ifnar1*^_KO n=8; IFNα-*Itgax^Ifnar1*^_KO n=8 and NaCl-*VeCad^Ifnar1*^_KO n=10; IFNα-*VeCad^Ifnar1*^_KO n=9. Data pooled from at least two independent experiments of each experimental group; p-values were calculated by log-rank/Mantel-Cox test.

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Next, we crossed Ifnar1-floxed mice (termed Ifnar1^fl/fl) (Prigge et al., 2015) with transgenic mice selectively expressing Cre recombinase in parenchymal and non-parenchymal liver cells (Gerl et al., 2015; Postic et al., 1999; Wang et al., 2010; Figure 4—figure supplement 1D). Cell type-specific recombination was confirmed by crossing each parental mouse line with Rosa26-ZsGreen reporter mice. Note that by crossing the parental Cre-expressing lines with Rosa26-ZsGreen reporter mice (Madisen et al., 2010) the resultant mice showed specific recombination in most hepatocytes (identified by morphology), and liver fibroblast (GFP⁺/PDGFRβ⁺), with about 98.2 ± 0.72% hepatic stellate cells that co-expressed GFP⁺ and PDGFRβ⁺ signals (Figure 4—figure supplement 1E, F). Similarly, hepatic DCs (GFP⁺/CD11c⁺) had 94.17 ± 2.16% colocalization with GFP, while the colocalization percentage of F4/80⁺ KCs or LCMs (GFP⁺/F4/80⁺) was 78.14 ± 5.03% (Figure 4—figure supplement 1E,F; Blériot and Ginhoux, 2019; Karmaus and Chi, 2014; Madisen et al., 2010). Finally, HECs, including LSECs, (GFP⁺/CD31⁺) showed 85.3 ± 5.03% colocalization (Figure 4—figure supplement 1E, F), with no expression of GFP signals in cells other than CD31⁺. Ifnar1^fl/fl control mice and mice lacking Ifnar1 in hepatocytes (termed Alb^Ifnar1^_KO), hepatic stellate cells (termed Pdgfrb^Ifnar1^_KO), Itgax⁺ (CD11c) DCs/KCs/LCMs (termed Itgax^Ifnar1^_KO) or Cdh5⁺ + cells (termed VeCad^Ifnar1^_KO) were intramesenterically injected with MC38 cells 7 days after NaCl or IFNα therapy initiation and did not show signs of hematotoxicity during IFNα infusion (Figure 4—figure supplement 2B). Metastatic growth was assessed by MRI at day 21 (Figure 4B). Loss of Ifnar1 on hepatocytes, hepatic stellate cells or DCs/KCs/LCMs did not significantly alter the anti-metastatic activity of IFNα treatment (Figure 4C, D and F and Figure 4—figure supplement 2C). By contrast, the depletion of Ifnar1 on HECs allowed the lesions to grow undisturbed (Figure 4B). Indeed, VeCad^Ifnar1^_KO mice treated with either NaCl or IFNα displayed very similar numbers and sizes of hepatic lesions (Figure 4C and D) or survival rates (Figure 4F), indicating that the antimetastatic properties of IFNα requires Ifnar1 signaling on HECs. VeCad^Ifnar1^_KO mice exhibited increased tumor burden (Figure 4D) and mortality rates (Figure 4—figure supplement 2D) when compared to NaCl-treated Ifnar1^fl/fl mice, suggesting that hepatic endothelial Ifnar1 signaling exerts significant anti-tumor activity even in the context of physiologic endogenous intrahepatic levels of type I interferons. Furthermore, histological analysis of hepatic CRC lesions from NaCl- and IFNα-treated VeCad^Ifnar1^_KO mice euthanized at day 21 after MC38 intramesenteric injection indicated that these tumors resembled NaCl-treated Ifnar1^fl/fl lesions, showing signs of angiogenesis (as denoted by CD34 positivity) and similar content of F4/80⁺ macrophages and CD3⁺ T cells within the intrahepatic CRC foci (Figure 4—figure supplement 3A, B).

Continuous IFNα administration limits trans-sinusoidal migration of CRC cells by strengthening the liver vascular barrier

We next took advantage of fluorescence-based techniques to investigate the initial steps of liver colonization. First, we assessed the intrahepatic localization of GFP-expressing MC38 cells (MC38^GFP) (Talamini et al., 2021) that were intramesenterically challenged 5 min earlier. Most MC38^GFP cells in Ifnar1^fl/fl or VeCad^Ifnar1^_KO mice appeared physically trapped at the beginning of the sinusoidal circulation in both mouse lineages. This was evidenced by the close contact of MC38^GFP cells with LYVE-1-expressing LSECs in the proximity of the portal tracts (Figure 5—figure supplement 1A). Further, MC38^GFP cells arrested where the sinusoidal diameter (Figure 5—figure supplement 1B) is smaller than their own (12±0.1 µm), similar to what we previously reported (Catarinella et al., 2016). As the process of trans-sinusoidal migration – a critical limiting step in the metastatic cascade – is known to occur within 24 hr of CRC challenge (Chambers et al., 2002; Valastyan and Weinberg, 2011; Wolf et al., 2012), the intrahepatic number and localization of MC38^GFP cells were then studied at this time point. Confocal IF quantification revealed that, compared to NaCl-treated Ifnar1^fl/fl animals, MC38^GFP cells were about ~twofold less abundant in IFNα-treated Ifnar1^fl/fl mice and ~threefold more abundant in VeCad^Ifnar1^_KO mice treated with NaCl or IFNα (Figure 5A top). Moreover, confocal 3D reconstructions of liver sinusoids from IFNα-treated Ifnar1^fl/fl mice unveiled that by 24 hr most MC38^GFP cells localize intravascularly (i.e. they did not invade the liver parenchyma), while in NaCl-treated Ifnar1^fl/fl controls and in NaCl- or IFNα-treated VeCad^Ifnar1^_KO mice only few MC38^GFP cells remain within the liver vasculature (i.e. they invaded the liver parenchyma) (Figure 5A bottom, 5B and Figure 5—videos 1–4). These results indicate that HECs, including LSECs, negatively control trans-sinusoidal CRC migration upon IFNα sensing.

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Continuous IFNα administration limits trans-sinusoidal migration of CRC cells by strengthening the hepatic vascular barrier.

(A) Total number of MC38^GFP cells per area (top) and total number of intravascular MC38^GFP cells per tissue area (bottom). The total hepatic area was approximately 5 mm² for each experimental group. Intravascular localization was measured on 20 x images, 5 randomly selected images per mouse (n=3 per group). Mean values ± SEM are shown; p-values were calculated by Mann-Whitney test. (B) Confocal reconstruction of the liver vasculature from *Ifnar1^fl/fl* and *VeCad^Ifnar1*^_KO mice 24 hr after MC38^GFP cells (green) intramesenteric vein injection in mice that were treated with NaCl or IFNα for 7 days. CD31 is shown in grey. To allow visualization of intravascular MC38^GFP cells and to enhance image clarity, the transparency of the sinusoidal rendering was increased up to 80% (bottom). Scale bars = 20µm. (C) Fenestrae frequency histogram (top) and percentage of vessel porosity (the percentage of liver endothelial surface area occupied by fenestrae, bottom) in liver sections of control *Ifnar1^fl/fl* and *VeCad^Ifnar1*^-KO mice treated for 7 days with continuous NaCl or IFNα therapy. Quantification was performed on 17.000 x SEM images, 10 randomly selected images per mouse (n=3 per group). A total area of approximately 720 µm² of sinusoidal surface was analyzed for each mouse. Mean values ± SEM are shown; p-values were calculated by Mann-Whitney test. (D) Representative scanning electron micrographs (SEM, top) and transmission electron micrographs (TEM, bottom) images from liver sections of mice described in (C), showing hepatic fenestrations and endothelial features. Arrow indicates the increased endothelial thickness observed after continuous IFNα therapy in *Ifnar1^fl/fl* mice. SEM scale bars = 1 µm; TEM scale bars = 500 nm. (E) Quantification of the arithmetical mean deviation or Ra coefficient (top) and the percentage of hepatic area positive for HS staining (bottom) on *Ifnar1^fl/fl* and *VeCad^Ifnar1*^_KO livers treated with continuous NaCl or IFNα therapy for 7 days. Quantification was performed on at least 3 *Ifnar1*^fl/fl and *VeCad^Ifnar1*^_KO livers per group. Mean values ± SEM are shown; p-values were calculated by Mann-Whitney test. (F) Liver sinusoidal endothelial glycocalyx (GCX) visualization by scanning electron micrographs (SEM, top) from *Ifnar1^fl/fl* and *VeCad^Ifnar1*^_KO livers that were perfused with lanthanum nitrate (a heavy metal that allows GCX visualization by stabilizing negatively charged GCX structures) 7 days after continuous NaCl or IFNα therapy. Scale bars = 1 µm. Inserts display a representation of the 3D topographic surface of a selected area within the liver sinusoid of each experimental condition. Representative immunofluorescence micrographs of Heparan sulfate (HS; green) and CD31 (red) staining from *Ifnar1^fl/fl* and *VeCad^Ifnar1_KO* mice treated with continuous NaCl or IFNα therapy for 7 days, showing increased HS accumulation after IFNα therapy only in *Ifnar1^fl/fl* mice (bottom). Hoechst (blue) was used for nuclear counterstaining. Scale bars = 20µm.

Figure 5—source data 1 Data for the graphs in the figure.: https://cdn.elifesciences.org/articles/80690/elife-80690-fig5-data1-v1.zip
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Figure 5—source data 2 High-magnification immunofluorescence images of each channel.: https://cdn.elifesciences.org/articles/80690/elife-80690-fig5-data2-v1.zip
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To unravel phenotypic modifications associated with such antitumor function of HECs, including LSECs, the liver microvasculature of NaCl- or IFNα-treated Ifnar1^fl/fl and VeCad^Ifnar1_KO mice was analyzed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). IFNα treatment of Ifnar1^fl/fl mice significantly decreased the frequency of sinusoidal fenestrae and the overall porosity of LSECs (Figure 5C and D top), while it increased: (i) the endothelial thickness (Figure 5—figure supplement 1D), (ii) the space of Disse density (an indirect measure of hepatocyte microvilli density) (Figure 5—figure supplement 1D; Gissen and Arias, 2015), (iii) the subendothelial deposition of collagen fibrils (Figure 5—figure supplement 1D) and (iv) the appearance of a basal lamina (Figure 5D bottom and Figure 5—figure supplement 1D). These results were corroborated by immunofluorescence analysis assessing an enhanced perivascular expression of Collagen type IV and Laminin (Figure 5—figure supplement 1E, F), two components of the basal lamina previously shown to form a barrier against tumor cell invasion (Mak and Mei, 2017; Tanjore and Kalluri, 2006). By contrast, IFNα treatment of VeCad^Ifnar1^_KO mice failed to significantly support these changes, leaving the liver microvasculature of these animals highly similar to that of liver metastases-permissive NaCl-treated Ifnar1^fl/fl controls (Figure 5C and D and Figure 5—figure supplement 1D-F). Moreover, IFNα treatment of Ifnar1^fl/fl mice significantly increased the expression of LYVE-1, a marker of hepatic capillarization (Pandey et al., 2020; Wohlfeil et al., 2019). By contrast, IFNα treatment of VeCad^Ifnar1^_KO mice showed no effect (Figure 5—figure supplement 2A,B).

Next, we evaluated the status of the vascular glycocalyx (GCX), a fibrous network of glycoproteins and proteoglycans that lines the LSECs and projects intraluminally (Reitsma et al., 2007). Notably, enhanced GCX deposits can act as a repulsive barrier that prevents tumor cell interactions with endothelial cells, adhesion molecules or chemokines have been previously identified as negative correlates of transendothelial migration (Glinskii et al., 2005; Mitchell and King, 2014; Offeddu et al., 2021; Wilkinson et al., 2020). Continuous IFNα treatment modified this network as well, increasing its thickness (Figure 5E and F top) and the expression of one of its major components, the heparan sulfate (HS) (Reitsma et al., 2007; Figure 5E and F bottom). Of note, VeCad^Ifnar1^_KO mice displayed reduced GCX thickness independently of NaCl- or IFNα-treatment (Figure 5E and F). Additionally, we evaluated the vascular and perivascular status of cell adhesion molecules such as selectins and integrins, which have been positively associated with the transendothelial migration of tumor cells (Glinskii et al., 2005; Wilkinson et al., 2020). The expression of ICAM1, E-Selectin (CD62E) (Figure 5—figure supplement 1G,H) and the integrins ITGB2 (CD18) or ITGA4 (CD49d) (Figure 5—figure supplement 1C) was up-regulated in IFNα-treated Ifnar1^fl/fl controls, while significantly reduced or attenuated in IFNα-treated VeCad^Ifnar1_KO mice. The notion that a more modest upregulation of some these markers was still evident in the latter mice may reflect the capacity of liver cells other than HECs to respond to IFNα. Altogether, these results indicate that numerous phenotypic modifications of the liver microvasculature previously associated with the deficient extravasation of both normal and transformed cells of different origin (Guidotti et al., 2015; Valastyan and Weinberg, 2011) also occur because of continuous IFNα sensing by HECs, including LSECs. Notably, these microvascular modifications were reverted after the discontinuation of IFNα therapy with no impact on long-term liver functionality/viability (Figure 5—figure supplement 2C-I).

HECs acquire an antimetastatic transcriptional profile upon continuous IFNα sensing

To confirm the above-mentioned data and to shed new light on the transcriptional changes that HECs adopt to limit CRC trans-sinusoidal migration, we performed RNA-seq analyses on CD31⁺ endothelial cells isolated from the liver of Ifnar1^fl/fl or VeCad^Ifnar1_KO mice 7 days after NaCl or IFNα treatment (Figure 6—figure supplement 1A). Using SEM to assess the % of CD31⁺ cells bearing the typical sinusoidal fenestrae, we determined that our preparations contain ~96% of bona-fide LSECs (Figure 6—figure supplement 1B), consistently to previous reports (Liu et al., 2011; Su et al., 2021). When compared to HECs isolated from NaCl-treated Ifnar1^fl/fl mice, HECs derived from IFNα-treated animals of the same lineage showed 381 transcripts that were differentially expressed (Figure 6A). As expected, many of these up-regulated transcripts belonged to the ISG family, including Irf7, Irf9, Mx1, Mx2, Isg15, Stat1, and Oasl1 (Figure 6A and B). Pre-ranked gene set enrichment analyses (GSEA) of IFNα-treated LSECs also revealed a significant enrichment of transcripts involved in interferon signaling or in the induction of varying cytokines and chemokines (Figure 6C). Several transcripts related to the ECM/GCX organization or the cell-cell/cell-matrix adhesion pathways were upregulated as well (Figure 6B and D). Of note, the expression of Itga4 and Itgb2 – previously shown to be increased by IFNα treatment at the protein level (Figure 5—figure supplement 1C) – was also enhanced at the transcriptional level (Figure 6B). A similar association did not hold true for Icam1 and Sele, suggesting that the increased protein expression we observed earlier (Figure 5—figure supplement 1G) occurred independently of transcriptional activity (Figure 6B). Notably, GSEA also identified gene sets involved in the IFNα-dependent activation of innate and adaptive immune responses or in TCR-dependent signaling pathways (Figure 6—figure supplement 1C).

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HECs acquire an antimetastatic transcriptional profile upon continuous IFNα sensing.

(A) Volcano plots of differential gene expression (DGE) results obtained from the comparisons between HECs-derived from *Ifnar1^fl/fl* mice treated with NaCl and IFNα for 7 days (left) and between HECs-derived from *VeCad^Ifnar1*^_KO and *Ifnar1^fl/fl* NaCl-treated mice (right) (*Ifnar1^fl/fl* n=3; *VeCad^Ifnar1*^_KO n=4). Significant false discovery rate (FDR <0.05) up- and down-regulated genes are highlighted in red and blue colors, respectively, not significant genes are depicted in grey. (B) Heatmap of the expression values (log2-transformed rpkm) of manually selected genes retrieved from differentially regulated pathways. (C) Pre-ranked Gene Set Enrichment Analysis (GSEA) enrichment plots of the indicated pathways between CD31⁺ cells from *Ifnar1^fl/fl*-IFNα and *Ifnar1^fl/fl*-NaCl (left) and between *VeCad^Ifnar1*^_KO-NaCl and *Ifnar1^fl/fl*-NaCl-treated animals (right). (D) Bar charts showing the adjusted p-values (-log10 transformed) of selected pathways from the enrichment analysis performed on comparisons between *Ifnar1^fl/fl*-IFNα and *Ifnar1^fl/fl*-NaCl CD31⁺ cells (left graph) and between *VeCad^Ifnar1*^_KO-NaCl and *Ifnar1^fl/fl*-NaCl CD31⁺ cells (right graph).

Figure 6—source data 1 Gene Ontology (GO) of biological process enrichment analysis of all differentially expressed genes. Excel spreadsheet with all significant pathways found in the comparison between IFNα-treated Ifnar1^fl/fl vs NaCl-treated Ifnar1^fl/fl HECs/LSECs and NaCl-treated VeCad^Ifnar1_KO-NaCl vs NaCl-treated Ifnar1^fl/fl LSECs were generated using GO Biological Processes 2018 collection.: https://cdn.elifesciences.org/articles/80690/elife-80690-fig6-data1-v1.zip
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Keeping the HECs transcriptional profile of NaCl-treated Ifnar1^fl/fl mice as a point of reference, a total of 566 genes were differentially expressed (DEGs) in HECs isolated from NaCl-treated VeCad^Ifnar1^_KO mice, of which 373 (mostly ISGs and genes involved in the immune response or in the antigen processing) were downregulated (Figure 6A–C). These latter results indirectly suggest that – when compared to HECs capable of sensing low levels of endogenous type I IFNs, as those present in NaCl-treated Ifnar1^fl/fl mice – LSECs devoted of Ifnar1 may be less prepared to stimulate innate and adaptive immunity (Figure 6B). The downregulation of transcripts involved in cell-cell adhesion molecules and matrix remodeling (Figure 6—figure supplement 1D) further suggests a relative unpreparedness of VeCad^Ifnar1^_KO HECs at accommodating changes that may confer protection against CRC trans-sinusoidal cell migration. Along these lines, the upregulation of the transcripts for Gata4 -a master-regulator of liver sinusoidal differentiation which leads to liver fibrosis deposition upon its loss (Winkler et al., 2021)- and Smad7 – an inhibitor of transforming growth factor-beta (TGF-β)-dependent subendothelial matrix deposition causative of sinusoidal capillarization (Tauriello et al., 2018) – in HECs isolated from NaCl-treated VeCad^Ifnar1^_KO mice (Figure 6A) could be interpreted as a diminished capacity to shape a vascular antimetastatic barrier. Finally, Gene Ontology (GO) analysis of HECs confirmed that Ifnar1-proficient, but not Ifnar1-deficient, HECs upregulate transcriptional pathways involved in the production of immunostimulatory cytokines and chemokines, the capacity to process and present antigens or the regulation of immune responses (Figure 6D). Altogether, the data support the hypothesis that, upon IFNα sensing, HECs and particularly LSECs not only acquire a transcriptional profile that can reinforce their barrier function, but they may also enhance HECs/LSECs immunostimulatory functions contributing to antitumor activity.

Continuous IFNα sensing improves immunostimulatory properties of HECs to provide long-term tumor protection

First, HECs/LSECs isolated from the liver of Ifnar1^fl/fl or VeCad^Ifnar1^_KO mice 7 days after continuous NaCl or IFNα treatment were assessed for the relative surface protein expression of MHC-I, CD86 (a costimulatory molecule Katz et al., 2004) or the interleukin 6 receptor alpha (IL-6RA, a molecule that LSECs use to properly cross-prime antigens to naïve CD8⁺ T cells Böttcher et al., 2014). Following IFNα treatment, Ifnar1-bearing LSECs significantly increased MHC-I, CD86 and IL-6RA expression (Figure 7—figure supplement 1A), while no induction was detected in Ifnar1-negative LSECs (Figure 7—figure supplement 1A). We then analyzed the ability of IFNα-treated LSECs or splenic DCs (sDCs) from Ifnar1^fl/fl and VeCad^Ifnar1^_KO mice to stimulate the cross-priming of naive CD8⁺ T cells in vitro. To this end, viable CD31⁺ HECs and CD11c⁺ sDCs were isolated and purified (Figure 7—figure supplement 2A, B). sDCs were cultured to acquire mature CD8α⁺ (~25%) or plasmacytoid (45%–50%) phenotypes endowed with cross-priming capacity (Figure 7—figure supplement 2C, D; Fu et al., 2020). HECs and sDCs from Ifnar1^fl/fl or VeCad^Ifnar1^_KO mice previously pulsed with the SIINFEKL peptide or soluble ovalbumin (sOVA) in the presence or absence of either IFNα or NaCl were then co-cultured with naive OT-I CD8⁺ T cells and their relative cross-priming capacity was defined by the percentage of these latter cells to express both CD44 and IFNγ (Figure 7—figure supplement 1B). IFNα stimulation of Ifnar1-bearing HECs (HECs from Ifnar1^fl/fl mice or sDCs from Ifnar1^fl/fl and VeCad^Ifnar1^_KO mice) pulsed with SIINFEKL or sOVA promptly increased their cross-priming capacities, while the same IFNα treatment failed to do so in Ifnar1-negative cells (HECs from VeCad^Ifnar1^_KO mice) (Figure 7A and B and Figure 7—figure supplement 1B, C). Once exposed to IFNα and pulsed with sOVA, HECs and sDCs from Ifnar1^fl/fl mice cross-primed naïve OT-I CD8⁺T cells to a similar extent (Figure 7B and Figure 7—figure supplement 1C), highlighting once more the immunostimulating potential of IFNα treatment on HECs, including LSECs. We also evaluated the splenic composition of central memory T cell populations (Tcm, CD8⁺CD44⁺CD62L⁺) (Figure 7C and Figure 7—figure supplement 1D) as a proxy of potential systemic memory responses against tumor antigens (Sallusto et al., 2004; Stone et al., 2009; Yu et al., 2019). Splenic naive T cells (Tn, CD8⁺CD44^-CD62L⁺) were also evaluated. Looking at Ifnar1^fl/fl or VeCad^Ifnar1^_KO mice continuously treated with NaCl or IFNα and euthanized by day 21 after challenge, we found that only IFNα-treated Ifnar1^fl/fl mice showed an increased proportion of Tcm and decreased percentage of Tn when compared to NaCl-treated Ifnar1^fl/fl controls (Figure 7C), suggesting that IFNα-responsive LSECs may promote antitumor immune memory in secondary lymphoid organs.

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Continuous IFNα sensing improves immunostimulatory properties of HECs to provide long-term tumor protection.

(**A–B**) Quantification of the percentage of OT-I CD8⁺ T cells expressing CD44⁺IFNγ⁺ generated after the co-cultured with HECs, including LSECs, or sDCs isolated from *Ifnar1^fl/fl* and *VeCad^Ifnar1*^-KO mice and stimulated with SIINFEKL peptide (A) or soluble OVA protein (sOVA) (B) in the presence of NaCl or IFNα. Mean values ± SEM are shown; p-values were calculated by two-way ANOVA test from two independent experiments with three biological replicates each. (C) Quantification of the percentage of splenic T central memory (Tcm, CD8⁺CD44⁺CD62L⁺) and naïve T cells (Tn, CD8⁺CD44^-CD62L⁺) populations 21 days after intramesenteric MC38 cells injection into NaCl- or IFNα-treated *Ifnar1^fl/fl* and *VeCad^Ifnar1*^_KO mice. Quantification was performed on least 4 mice per group. Mean values ± SEM are shown; p-values were calculated by Mann-Whitney test. (D) Schematic representation of the experimental procedure used for tumor rechallenge. IFNα-*Ifnar1^fl/fl*-cured mice and naïve *Ifnar1^fl/fl* mice were subcutaneously rechallenged with 5x10³ MC38 cells and received no further treatment. Tumor growth was monitored weekly for 35 days. Note that we used aged-matched *Ifnar1^fl/fl* naïve mice because NaCl-treated mice had to be euthanized for ethical reasons by day 50. (E) Kinetics of subcutaneous tumor growth in naïve (n=10) and IFNα-cured (n=7) mice. Both, mean tumor volume and individual animal measurements are shown. Mean values ± SEM are shown; p-values were calculated by two-way ANOVA test. (F) Schematic model: CRC cells emerging from the primary tumor reach the hepatic sinusoids via the portal circulation and arrest - mostly because of size constrains - at the portal side of the sinusoidal circulation (1), CRC cells trans-sinusoidally migrate into the liver parenchyma (2) and develop micro-metastases that will eventually grow overtime (3; red frame), promoting the generation of an immunosuppressive microenvironment leading to dysfunctional T cells (4; red frame). Conversely, IFNα therapy (blue frame), by modifying LSECs porosity, thickness, deposition of basal membrane and GCX depth, builds up a vascular antimetastatic barrier (5), that impairs CRC trans-sinusoidal migration, promoting intravascular containment of invading tumor cells (6) that together with IFNα-mediated increased cross-presentation and cross-priming by HECs/LSEC, will lead to naïve CD8⁺ T cell activation and secondary generation of long-term antitumor immunity and protection from secondary tumor challenge (7). Created with https://biorender.com/.

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To assess whether IFNα-stimulated HECs and LSECs promoted memory responses endowed with antitumor potential, Ifnar1^fl/fl-cured mice (defined as animals that 7 days after IFNα treatment initiation were intramesenterically challenged with MC38 cells and survived as disease-free animals until day 50) or naive Ifnar1^fl/fl control mice were subcutaneously rechallenged with MC38 cells (Figure 7D). Notably, while the latter animals developed subcutaneous tumors that increased in size over time, none of the Ifnar1^fl/fl-cured mice showed detectable lesions at any time point studied (Figure 7E). These results indicate that continuous IFNα treatment promotes protection against secondary tumor challenge even after IFNα therapy discontinuation. The results also suggest that this effect may be dependent on the capacity of IFN-sensitive HECs and LSECs to foster antitumor immunity, especially tumor-specific effector CD8⁺ T cell responses that are well-known to control tumor growth in vivo in different experimental settings (Dobrzanski et al., 2000; Katlinski et al., 2017; Klebanoff et al., 2005; Yu et al., 2019).

Discussion

In this study, we used different mouse models of CRC liver metastasis to show that the continuous perioperative administration of relatively low IFNα doses provides significant antitumor potential in vivo without provoking overt toxicity. Moreover, under the pharmacological conditions we defined (route, dosage, treatment duration, and chemical nature of the recombinant protein), we did not observe counter-regulatory mechanisms affecting IFNα efficacy (Katlinski et al., 2017), or significant systemic side effects, as our strategy avoids the short tissue-oscillatory IFNα bursts that are often achieved after high and pulsed administrations, often associated with efficacy-limiting toxicities (Weber et al., 2015). These results are consistent with previous preclinical work indicating that the intrahepatic delivery of IFNα through a gene/cell therapy approach curbs CRC liver metastases by acting primarily on unidentified non-hematopoietic stromal cell populations (Catarinella et al., 2016).

Given the pleotropic nature of IFNα, we demonstrated that the antimetastatic activity of IFNα is neither based on the direct inhibition of primary intracecal tumor growth, favoring the hypothesis that IFNα therapy does not modify the number of cells that spread from primary tumors and seed into the liver – nor on the direct inhibition of metastatic cell growth within the liver. These data are consistent with the high IFNα concentrations required to activate the ‘tunable’ direct antiproliferative functions of this cytokine, likely exceeding the levels achieved in our system (Catarinella et al., 2016; Schreiber, 2017). In addition, IFNα therapy does not require indirect stimulation of hepatocytes, HSCs, DCs, KCs, or LCMs to exert its antimetastatic functions. Rather, the results pinpointed HECs/LSECs as key local and early sensors of IFNα that ultimately limit CRC cell invasion into the liver.

Mechanistically, we showed that IFNα-stimulated LSECs inhibit the trans-sinusoidal migration of circulating CRC cells normally occurring within 24 hr of their initial intrahepatic landing. This effect is associated with phenotypic changes that IFNα-stimulated LSECs acquire or induce in the liver microenvironment. Among these changes, we observed a reduction in the overall LSEC porosity (i.e. sinusoidal fenestrae were reduced in number and size), an enhancement in the subendothelial deposition of basal membrane components (including Collagen IV and Laminin) and an upregulation of LYVE-1, a marker of hepatic capillarization (Pandey et al., 2020; Wohlfeil et al., 2019). Along these lines, it is noteworthy that in the ‘healthy’ liver, functioning as a common site for CRC metastases, LSECs contain numerous fenestrae of up to 200 nm in diameter and normally lack the typical basal membrane that characterizes the microvasculature of most other tissues and organs (Jacobs et al., 2010). It is also interesting to note that IFNα-stimulated LSECs promote microvascular alterations like those typifying pathological conditions (e.g. initial hepatic capillarization and liver fibrosis Pandey et al., 2020; Wohlfeil et al., 2019) associated with impaired immune cell extravasation and reduced immune surveillance (Guidotti et al., 2015) and reduction of hepatic metastases from solid tumors including CRCs (Wohlfeil et al., 2019). This fits with the evidence that CRC patients suffering from chronic viral liver fibrotic diseases characterized by hepatic endogenous type I interferon production display lower incidence of hepatic metastases (Augustin et al., 2013; Baiocchini et al., 2019; Li Destri et al., 2013). The existence that fibrotic liver diseases not associated with reduced metastatic risk (Kondo et al., 2016) suggests that changes in the vascular hepatic niche other than matrix deposition play additional roles in this process. Indeed, IFNα stimulated LSEC-governed changes hampering CRC extravasation including the modification of the sinusoidal GCX that, by increasing its thickness and modifying its chemical composition, recapitulated conditions known to negatively regulate the trans-endothelial migration of tumor cells in other settings (Glinskii et al., 2005; Mitchell and King, 2014; Offeddu et al., 2021; Wilkinson et al., 2020). The continuous administration of therapeutic low-doses of IFNα thus stimulate HECs/LSECs to shape a vascular antimetastatic barrier preventing the interaction between tumor cells and endothelial cells that are known to promote the extravasation of the former cells (Glinskii et al., 2005; Mitchell and King, 2014; Wilkinson et al., 2020). Accordingly, the enhanced expression of ‘pro-migratory’ adhesion molecules and integrins that we observed in the liver of animals bearing IFNα-responsive LSECs appear to be efficiently counteracted by the creation of such vascular barrier.

Of note, the functional consequences of LSEC capillarization (especially the induction of hepatic fibrosis) during states of chronic liver injury highly depend on the relative magnitude and duration of the underlying liver disease (DeLeve, 2015). Additionally, both LSEC capillarization and hepatic fibrosis are reversed when chronic liver injury resolves (DeLeve, 2015). In keeping with these concepts and the absence of sALT elevation or morphological evidence of liver disease during continuous IFNα therapy, it is not surprising that we observed a complete recovery of fenestrae abundance and LSEC porosity 40 days after therapy discontinuation. This supports the notion that a continuous but relatively short IFNα therapy promotes changes in the structure and function of LSECs that are mild and reversible and should not result in persistent hepatic fibrinogenesis. Such notion is also supported by the absence of hepatic toxicity (Weber et al., 2015) and the significant reduction in established fibrosis in patients with chronic viral liver diseases treated with recombinant IFNα for up to 48 weeks (Li et al., 2019; Poynard et al., 2002). The notion that IFNα treatment failed to shape the vascular antimetastatic barrier in mice carrying the Ifnar1-deficiency only in endothelial cells further strengthens this hypothesis and places HECs/LSECs at a center of a relevant antitumor process ultimately limiting CRC liver invasion. This concept is also indirectly supported by the fact that the increased expression Gata4 -a master-regulator of liver sinusoidal differentiation which leads to liver fibrosis deposition upon its loss (Winkler et al., 2021) - and Smad7 – an inhibitor of transforming growth factor-beta (TGF-β) dependent subendothelial matrix deposition and sinusoidal capillarization (Tauriello et al., 2018) were not downregulated by IFNα treatment in mice in which all cell types except LSECs could sense this cytokine, thus loosening the hepatic vascular barrier.

In addition, to hindering the initial trans-sinusoidal migration of CRC cells in vivo, IFNα-stimulated LSECs efficiently cross-presented nominal tumor antigens to naive CD8⁺ T cells in vitro, enabling degrees of T cell priming and effector differentiation that were comparable to those induced by professional APCs. In keeping with this, we demonstrated that the in vivo IFNα stimulation of LSECs resulted in the upregulation of proteins and transcripts associated with antigen processing and presentation or co-stimulation (e.g. MHC-I, CD86, IL-6RA, B2m, Tap1, Psmb-8/9 and H2-d1, H2-k1/2) (Böttcher et al., 2014; Katz et al., 2004; Montoya et al., 2002; Rodriguez et al., 1999). Moreover, our results also suggest that IFNα-stimulated LSECs may play a key role in antitumor immunity, as mice were protected from secondary tumor rechallenge even after discontinuation of IFNα treatment. The fact that the same IFNα therapy also significantly increased the overall number of central memory T cells in the spleen while decreasing that of naive T cells (Sallusto et al., 2004; Yu et al., 2019) further suggests a role for IFNα-stimulated LSECs in the generation of systemic and protective long-term antitumor immunity.

These data are consistent with the notion that IFNα-stimulated LSECs, due to their anatomical proximity and efficient endocytosis capacity that is among the highest of all cell types in the body (Sorensen and Smedsrod, 2020) – rapidly remove CRC-derived antigens from the intravascular space and productively and rapidly contribute to the development of effective antitumor immunity, since this process does not require the time-consuming step of migration to lymphatic tissue (Böttcher et al., 2014). This concept is also supported by the upregulation by IFNα-stimulated LSECs of Cxcl9 and Cxcl10, two chemokines involved in the attraction and retention of naïve T cell populations of lymphocytes into the liver (Franciszkiewicz et al., 2012), a necessary step for the generation of an efficient antitumor immune response. Additionally, other cell types within the hepatic niche could further amplify this IFNα-initiated cascade, as it has been shown that dendritic cells releasing IFNα also reduce liver metastatic colonization by CRC cells (Toyoshima et al., 2019) and that this cytokine properly polarizes the tumor microenvironment (Catarinella et al., 2016; De Palma et al., 2008). On the contrary, the notion that a minority of IFNα-treated animals develop small intrahepatic lesions that display similar proliferation, neoangiogenic and immunologic markers than untreated lesions highlights the possibility that CRC tumors, once established as macroscopic metastases, may become refractory and resistant to IFNα therapy by downregulating Ifnar1 (Boukhaled et al., 2021; Katlinski et al., 2017). This would be consistent with the lack of efficacy of our approach in established orthotopic tumors within the cecal wall.

Altogether, we have identified a novel MoA by which IFNα functions as antitumor drug against CRC liver metastases. Whether the adoption of similar LSEC-stimulating IFNα treatments may also curb the hepatic growth of metastatic cells originating from other solid tumors, or if continuous IFNα treatment promote the generation of vascular barriers in other metastasis-prone organs remains to be determined (Crist and Ghajar, 2021). Based on the findings of this report, we propose the following model: CRC cells emerging from the primary tumor reach the hepatic sinusoids via the portal circulation and arrest – mostly because of size constrains – at the portal side of the sinusoidal circulation. CRC cells then trans-sinusoidally migrate into the liver parenchyma and develop micrometastases that will eventually grow overtime, promoting the generation of an immunosuppressive microenvironment. Continuous therapy with well-tolerated doses of recombinant IFNα, stimulates HECs/LSECs to limit CRC trans-sinusoidal migration and parenchymal invasion by building up a vascular barrier typified by the reduction of LSECs porosity, the increased thickness of GCX and the appearance of a basal membrane. Continuous IFNα therapy also promotes long-term antitumor immunity in cured mice and protection from secondary tumor challenge, by stimulating LSECs to efficiently cross-prime tumor antigens to naïve CD8⁺ T cells (Figure 7F).

In terms of future clinical applications, our strategy could be used as perioperative neoadjuvant immunotherapy in CRC patients undergoing resection of their primary tumor who are at high risk for developing metachronous liver metastases (Engstrand et al., 2019; van Gestel et al., 2014). Indeed, several technologies have already been developed for the sustained release of drugs, such as osmotic pumps, electronic devices, hyaluronic acid-based hydrogels (Park et al., 2018; Stewart et al., 2018; Yun and Huang, 2016), FDA-approved polymer miscellas – such as pegylated (PEG)-IFNα (Foser et al., 2003; Glue et al., 2000) – and IFNα cell/gene therapy approaches (Catarinella et al., 2016), which could quickly translate our results into clinical practice. Of note, the use of clinically approved doses of pegylated-IFNα has shown improved serum stability and clinical efficacy and reduced side effects, with serum IFNα concentrations similar to those achieved in our system (Foser et al., 2003; Glue et al., 2000).

All in all, the results of this study support the use of continuous low doses of IFNα as an antimetastatic drug during the perioperative period, due to its ability to transform a metastases-prone liver into a metastases-resistant organ.

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Strain, strain background (Mus musculus)	C57BL/6 J	Charles River, Italy	Strain code: 632	Inbred
Strain, strain background (Mus musculus)	BALB/c	Charles River Lab	Strain code: 028	Inbred
Strain, strain background (Mus musculus)	NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ)	Charles River Lab	Strain code: 614	Immun odeficient
Strain, strain background (Mus musculus)	Ifnar1^fl/fl (B6(Cg)-Ifnar1tm1.1Ees/J)	Jackson Lab	JAX:028256	C57BL/6 J background
Strain, strain background (Mus musculus)	Alb^Cre (B6.Cg-Speer6-ps1Tg(Alb-cre)21Mgn/J)	Jackson Lab	JAX:003574	C57BL/6 J background
Strain, strain background (Mus musculus)	Pdgfrb^CreERT2 (B6.Cg-Tg(Pdgfrb-cre/ERT2)6096Rha/J)	Jackson Lab	JAX:029684	C57BL/6 J background
Strain, strain background (Mus musculus)	Itgax^Cre (CD11c, B6.Cg-Tg(Itgax-cre)1-1Reiz/J)	Jackson Lab	JAX:008068	C57BL/6 J background
Strain, strain background (Mus musculus)	Rosa26-ZsGreen (B6.Cg-Gt(ROSA)26Sortm6(CAG-ZsGreen1)Hze/J)	Jackson Lab	JAX:007906	C57BL/6 J background
Strain, strain background (Mus musculus)	VeCad^CreERT2 (Cdh5(PAC)^-CreERT2 mice)	Taconic, Models for life	Provided by S. Brunelli (UniMib, Milan)	C57BL/6 J background
Strain, strain background (Mus musculus)	OT-I mice (C57BL/6-Tg(TcraTcrb) 1100Mgb/Crl)	Charles River Lab	JAX:003831	C57BL/6 J background
Antibody	Anti-CD126, IL-6RA, (clone: D7715A7) (Rat monoclonal)	Biolegend	Cat# 115806, RRID:AB_313676	FC (1:100)
Antibody	anti-CD18, ITGB2 (clone M18/2) (Rat monoclonal)	Biolegend	Cat# 101402, RRID:AB_312811	FC (1:100)
Antibody	Anti-CD49d, ITGA4, (clone 9C10(MFR4.B)) (Rat monoclonal)	Biolegend	Cat# 103706, RRID:AB_313046	FC (1:100)
Antibody	Anti-H-2K^b/H-2D^b (clone 28-8-6) (Mouse monoclonal)	Biolegend	Cat# 114605, RRID:AB_313596	FC (1:100)
Antibody	Anti-CD86 (clone GL-1) (Rat monoclonal)	Biolegend	Cat# 105007, RRID:AB_313150	FC (1:100)
Antibody	Anti-CD146 (clone ME-9F1) (Rat monoclonal)	Biolegend	Cat# 134704, RRID:AB_2143527	FC (1:100)
Antibody	Anti-CD44 (clone IM7) (Rat monoclonal)	Biolegend	Cat# 103044, RRID:AB_2650923	FC (1:100)
Antibody	Anti-CD62L (clone MEL-14) (Rat monoclonal)	Biolegend	Cat# 104420, RRID:AB_493376	FC (1:100)
Antibody	Anti- CD16/CD32, (Rat monoclonal)	BioXCell	Cat# BE0307; RRID:AB_2736987	FC (1:100)
Antibody	Anti-GFP, (Rabbit polyclonal)	Thermo Fisher Sci	Cat# A11122, RRID:AB_221569	IF (1:100)
Antibody	Anti-GFP (clone FM264G) (Rat monoclonal)	Biolegend	Cat# 338008, RRID:AB_2563288	IF (1:100)
Antibody	Anti-CD31, PECAM1, (Goat polyclonal)	R&D Systems	Cat# AF3628, RRID:AB_2161028	IF (1:300)
Antibody	Anti-PDGFRβ, CD140b (clone APB5) (Rat monoclonal)	eBioscience	Cat# 14-1402-82, RRID:AB_467493	IF (1:200)
Antibody	Anti-CD11c, (clone N418)(Armenian hamster monoclonal)	Biolegend	Cat#117312, RRID:AB_492850	IF (1:100)
Antibody	Anti-Heparan Sulfate (clone F58-10E4) (Mouse monoclonal)	Amsbio	Cat# 370255 S, RRID:AB_10891554	IF (1:50)
Antibody	Anti-LYVE-1 (Rabbit polyclonal)	Novus Biol	Cat# NB600-1008, RRID:AB_10000497	IF (1:300)
Antibody	Anti-Collagen type IV (Rabbit polyclonal)	Abcam	Cat# ab19808, RRID:AB_445160	IF (1:100)
Antibody	Anti-Laminin (Rabbit polyclonal)	Sigma-Aldrich	Cat# L9393, RRID:AB_477163	IF (1:300)
Antibody	Anti-CD54, ICAM1 (clone YN1/1.7.4) (Rat monoclonal)	Biolegend	Cat#116101, RRID:AB_313692	IF (1:100)
Antibody	Anti-CD62E, E-selectin (clone 10E9.6) (Rat monoclonal)	BD Bioscience	Cat#550290, RRID:AB_393585	IF (1:100)
Antibody	Anti-Rabbit AF488 (Donkey polyclonal)	Thermo Fisher Sci	Cat#32790, RRID:AB_2762833	IF (1:200)
Antibody	Anti-Goat AF546 (Donkey polyclonal)	Thermo Fisher Sci	Cat#A-11056, RRID:AB_2534103	IF (1:200)
Antibody	Anti-Rat AF647 (Donkey polyclonal)	Jackson IR	Cat#712-605-153, RRID:AB_2340694	IF (1:200)
Antibody	Anti-IgM conjugated to APC (clone II/41) (Rat monoclonal)	Thermo Fisher Sci	Cat#17-5790-82, RRID:AB_469458	IF (1:100)
Antibody	Anti-Ki-67 (clone SP6) (Rabbit recombinant antibody)	Thermo Fisher Sci	Cat# MA5-14520, RID:AB_10979488	IHC (1:200)
Antibody	Anti-CD34 (clone MEC14.7) (Rat monoclonal)	Biolegend	Cat# 119301, RRID:AB_345279	IHC (1:300)
Antibody	Anti-F4/80 (clone A3-1) (Rat monoclonal)	Bio-Rad	Cat# MCA497, RRID:AB_2098196	IHC (1:200)
Antibody	Anti-CD3 (clone SP7) (Rabbit monoclonal)	Abcam	Cat# ab21703, RRID:AB_446487	IHC (1:100)
Antibody	Anti-pSTAT1 (Clone 58D6) (Rabbit monoclonal)	Cell Signaling	Cat# BK9167S, RRID:AB_561284	IHC (1:800)
Antibody	Anti-CD11c microbeads ultrapure	Miltenyi Biotec	Cat# 130-125-835	100 µL per 10⁸ total cells

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Selection of the optimal IFNα dosing regimen.

Figure 1—source data 1

Continuous IFNα administration reduces liver CRC metastatic burden and improves survival.

Figure 2—source data 1

Continuous IFNα administration prevents spontaneous hepatic colonization of orthotopically implanted CT26LM3 cells.

Figure 3—source data 1

HECs mediate the antimetastatic activity of IFNα.

Figure 4—source data 1

Continuous IFNα administration limits trans-sinusoidal migration of CRC cells by strengthening the hepatic vascular barrier.

Figure 5—source data 1

Figure 5—source data 2

HECs acquire an antimetastatic transcriptional profile upon continuous IFNα sensing.

Figure 6—source data 1

Continuous IFNα sensing improves immunostimulatory properties of HECs to provide long-term tumor protection.

Figure 7—source data 1

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Ngoc Lan Tran

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Lorena Maria Ferreira

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Blanca Alvarez-Moya

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Valentina Buttiglione

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Barbara Ferrini

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Paola Zordan

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Andrea Monestiroli

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Claudio Fagioli

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Eugenia Bezzecchi

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Giulia Maria Scotti

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Antonio Esposito

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Riccardo Leone

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Chiara Gnasso

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Andrea Brendolan

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Luca G Guidotti

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Giovanni Sitia

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Continuous IFNα administration prevents spontaneous hepatic colonization of orthotopically implanted CT26^LM3 cells.