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# Reprogramming Tumor-Associated Macrophages with Lipid Nanosystems Reduces PDAC Tumor Burden and Liver Metastasis

Adrián Palencia-Campos<sup>1,2</sup>, Laura Ruiz-Cañas<sup>1,2,3</sup>, Marcelina Abal-Sanisidro<sup>4,5,6</sup>, Juan Carlos López-Gil<sup>1,2,7</sup>, Sandra Batres-Ramos<sup>1,2</sup>, Sofia Mendes Saraiva<sup>4,6,8</sup>, Balbino Yagüe<sup>1,2</sup>, Diego Navarro<sup>1,2,7</sup>, Sonia Alcalá<sup>1,2</sup>, Juan A. Rubiolo<sup>9,10</sup>, Nadège Bidan<sup>11</sup>, Laura Sánchez<sup>9</sup>, Simona Mura<sup>11</sup>, Patrick C. Hermann<sup>12</sup>, María de la Fuente<sup>4,6,13</sup>, Bruno Sainz, Jr.<sup>1,2,6,†</sup>

<sup>1</sup>Cancer Stem Cells and Fibroinflammatory Microenvironment Group, Instituto de Investigaciones Biomédicas (IIBm) Sols-Morreale
CSIC-UAM, 28029 Madrid, Spain.

<sup>2</sup>Biomarkers and Personalized Approach to Cancer Group (BIOPAC), Area 3 Cancer, Instituto Ramón y Cajal de Investigación Sanitaria
(IRYCIS), 28049, Madrid, Spain.

<sup>3</sup>Biobanco Hospital Universitario Ramón y Cajal, IRYCIS, Madrid, Spain.

<sup>4</sup>Nano-Oncology and Translational Therapeutics group, IDIS, Complexo Hospitalario Universitario de Santiago de Compostela, 15706
Santiago de Compostela, Spain.

<sup>5</sup>University of Santiago de Compostela (USC), 15782 Santiago de Compostela, Spain.

<sup>6</sup>Centro de Investigación Biomédica en Red, Área Cáncer, CIBERONC, ISCIII, Madrid, Spain.

<sup>7</sup>Department of Biochemistry, Autónoma University of Madrid (UAM), 28029 Madrid, Spain.

<sup>8</sup>CPIRN-IPG- Center of Potential and Innovation of Natural Resources, Polytechnic Institute of Guarda, Av. Dr. Francisco de Sá
Carneiro, No. 50, 6300-559 Guarda, Portugal.

<sup>9</sup>Department of Zoology, Genetics and Physical Anthropology, Faculty of Veterinary, University of Santiago de Compostela (USC),
Lugo, Spain.

<sup>10</sup>Laboratorio Mixto de Biotecnología Acuática, Facultad de Ciencias Bioquímicas y Farmacéuticas, UNR, 2000, Rosario, Argentina.

<sup>11</sup>Université Paris-Saclay, CNRS, Institut Galien Paris-Saclay, 91400 Orsay, France

<sup>12</sup>Department of Internal Medicine I, Ulm University, Ulm, Germany

<sup>13</sup>DIVERSA Technologies S.L., Edificio Emprendia, Campus Sur, 15782 Santiago de Compostela, Spain.

<sup>†</sup>Correspondence: Bruno Sainz, Jr.: bsainz@iib.uam.es
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# ABSTRACT

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**Background:** Pancreatic ductal adenocarcinoma (PDAC) requires innovative therapeutic strategies to counteract its progression and metastatic potential. Since the majority of patients are diagnosed with advanced metastatic disease, treatment strategies targeting not only the primary tumor but also metastatic lesions are needed. Tumor-Associated Macrophages (TAMs) have emerged as central players, significantly influencing PDAC progression and metastasis. Our objective was to validate an innovative therapeutic strategy involving the reprogramming of TAMs using lipid nanosystems to prevent the formation of a pro-metastatic microenvironment in the liver.

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**Results:** *In vitro* results demonstrate that M2-polarized macrophages lose their M2-phenotype following treatment with lipid nanoemulsions composed of vitamin E and sphingomyelin (VitE:SM), transitioning to an M0/M1 state. Specifically, VitE:SM treatment decreased the expression of macrophage M2 markers such as *Arg1* and *Egr2*, while M1 markers such as *Cd86*, *Il-1b* and *Il-12b* increased. Additionally, the TGF-βR1 inhibitor Galunisertib (LY2157299) was loaded into nanoemulsions and delivered to C57BL/6 mice orthotopically injected with KPC PDAC tumor cells. Treated mice achieved diminished primary tumor growth and reduced TAM infiltration in the liver. Moreover, we observed a decrease in liver metastasis with the nanoemulsion treatment in an intrasplenic model of PDAC liver metastasis. Finally, we validated the translatability of our VitE:SM nanosystem therapy in a human cell-based 3D co-culture model *in vivo*, underscoring the pivotal role of macrophages in the nanosystem's therapeutic effect in the context of human PDAC metastasis.

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**Conclusions:** The demonstrated effectiveness and safety of our nanosystem therapy highlights a promising therapeutic approach for PDAC, showcasing its potential in reprogramming TAMs and mitigating the occurrence of liver metastasis.

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**KEYWORDS:** pancreatic ductal adenocarcinoma, tumor-associated macrophages, tumor microenvironment, lipid nanoemulsions, galunisertib, liver metastasis, reprogramming.

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BACKGROUND

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Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths in the United States¹, a consequence of various factors such as delayed diagnosis, the absence of specific markers and symptoms of early detection and the inherent metastatic nature of this tumor², ³. Notably, when PDAC is identified at an early stage, the 5-year overall survival rate following surgical resection of the primary tumor reaches 44%⁴; however, fewer than 20% of cases are eligible for surgical intervention⁵⁻⁷. Even in cases where the primary tumor is successfully resected, approximately 75% of patients develop metastatic relapse within five years post-resection⁸⁻¹⁰.

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The metastatic process of PDAC occurs through the bloodstream¹¹, with the liver being the primary target. Liver metastases are detected in 76-80%¹² of patients, limiting their eligibility for surgery¹³, ¹⁴. Additionally, metastases can also occur in the peritoneum (48%) and the lungs (45%), further complicating the management of PDAC patients¹². Since a substantial proportion of patients present with metastases at the time of diagnosis, it is imperative that we develop treatment strategies that target not only the primary tumor but also metastatic lesions.

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Metastatic dissemination in PDAC occurs through a series of orchestrated events¹⁵. PDAC cells release exosomes containing factors that are taken up by liver Kupffer cells, stimulating the production of fibrotic cytokines such as transforming growth factor-beta (TGF-β). TGF-β plays a crucial role in metastasis formation, participating in epithelial-mesenchymal transition (EMT), activation of cancer-associated fibroblasts (CAFs), and immune suppression¹⁶, ¹⁷. These cytokines stimulate hepatic stellate cells, prompting the synthesis and deposition of fibronectin, which establishes a fibrotic microenvironment in the liver. This environment fosters the recruitment of bone marrow-derived cells, including macrophages and neutrophils¹⁸, ¹⁹. These sequential processes culminate in the formation of a pre-metastatic niche²⁰, creating favorable conditions for the survival, proliferation, and subsequent metastatic growth of disseminated PDAC cells within the liver¹⁵.

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Within this dynamic metastatic tumor microenvironment, CAFs and immune cells, including macrophages, T cells, and neutrophils, create a niche for cancer cells to modulate tumor growth and their invasive behavior<sup>21-23</sup>. Tumor-associated macrophages (TAMs), in particular, have emerged as central orchestrators of PDAC tumorigenesis, significantly impacting the progression and metastasis of PDAC. Through interactions with PDAC cells, TAMs influence disease progression by facilitating resistance to chemotherapeutics, promoting metastasis and influencing cancer cell evasion of the antitumor immune response<sup>24-26</sup>. Macrophages, recognized for their plasticity, are broadly classified into M1 and M2 subtypes. M1 macrophages, characterized by high expression of CD86, IL-1β, and IL-12β, exhibit a pro-inflammatory profile and suppress tumor growth and progression<sup>27</sup>. On the other hand, M2 macrophages, characterized by high expression of CD206, ARG1, and CD163, promote tumor progression by antagonizing anti-tumor T-cell immunity<sup>28</sup>. TAMs often express an M2-like phenotype, which is associated with tumor-promoting activities<sup>29</sup>. The collaboration between PDAC cells and TAMs significantly contributes to disease progression, making TAMs an attractive target for alternative therapies<sup>30, 31</sup>. By shifting the polarization state of TAMs from the tumor-promoting M2 phenotype to the anti-tumoral M1 phenotype, one could potentially alter the tumor microenvironment, creating a hostile setting for cancer progression and preventing PDAC metastasis.

Nanotechnology is emerging as a promising tool in the biomedical field, with recent advancements focusing on reprogramming TAMs using nanomaterials, offering perspectives to inspire the design of new nanotechnology therapies to advance TAM-mediated immunotherapy<sup>32, 33</sup>. Specifically, organic nanoparticles are particularly attractive due to their translational potential, which can be attributed to their biocompatibility and biodegradability. Along these lines, nanoemulsions composed of vitamin E (VitE) and the biocompatible lipid sphingomyelin (SM) stand out as exceptionally promising carriers for delivering innovative therapeutics and advancing novel biomedical applications<sup>34</sup>, a trend observed across various cancer types<sup>35-37</sup>. These lipid nanosystems are readily phagocytosed by macrophages and can easily encapsulate therapeutics, such as potential macrophage reprogramming agents. Thus, they represent highly effective vehicles for targeted delivery to, and reprogramming of, TAMs. They have also proved to accumulate in the liver, being promising agents for treating liver metastasis<sup>38</sup>. In terms of composition, VitE has been
extensively utilized in numerous formulations intended for clinical use, due to its good biodegradability and
versatility in associating and encapsulating a variety of therapeutic molecules. It is also a potent antioxidant
and, moreover, recent discoveries highlight that VitE also has direct impacts on the immune system,
particularly on macrophages<sup>39-42</sup>. On the other hand, sphingolipids present a common structural feature, the
sphingosine backbone, which provides bioactive functions, from cell growth regulation to modulators of cell-
to-cell interactions<sup>43, 44</sup>. Specifically, SN is known to be a major component in lipid bilayers and, and serves as
a precursor to ceramide, a bioactive lipid molecule involved in cell signaling pathways that regulate
apoptosis, proliferation, and inflammation. SM role in modulating macrophage activation and resulting
inflammation has also been described<sup>45, 46</sup>.

The objective of this study was to test an innovative therapeutic approach involving reprogramming TAMs to
an M0/M1 state using a battery of lipid nanosystems, including VitE:SM nanoemulsions as a means of
inhibiting the formation of the pro-tumorigenic liver microenvironment that facilitates PDAC metastasis. This
targeted reprogramming holds promise not only in restraining the influence of TAMs on primary tumor
growth but also in mitigating their pro-metastatic impact in distant organs, particularly the liver. In addition,
our proposed approach capitalizes on the potential of these specific types of lipid nanosystems for the
precise and effective delivery of therapeutic agents already employed in the clinical settings to target the
pre-metastatic niche, such as the TGF-βR1 inhibitor Galunisertib<sup>47-49</sup>. This strategic and clinically-relevant
avenue of research offers a promising method for combating PDAC metastasis, reshaping the landscape of
pancreatic cancer treatment and offering hope and targeted solutions to patients.

## METHODS

**Lipid nanosystems preparation.** Nanosystems comprising VitE, SM, OA, M, PA, PI, PS, and PG lipids were formulated by adapting the straightforward and highly efficient ethanol injection method previously outlined by our group<sup>35, 37</sup> (Figure 1a). In short, various lipid compositions (Figure 1b, Table 1) were dissolved in 100 µL of 96% EtOH. Subsequently, nanosystems were promptly generated by injecting the organic phase into 1 mL of ultrapure water at room temperature (RT). The nanosystems were stored at 4°C until further use.
128 Formulation purification and concentration were achieved using spin filters (Amicon® Ultra Centrifugal
129 Filters, 100K, 0.5mL, Merck) in a refrigerated microcentrifuge (5417R; Eppendorf). The spin filters were
130 initially rinsed with ultrapure water at 6000×g for 5 min. The formulations were then transferred to the spin
131 filters and centrifuged for 5 min at 4°C and 3000×g. Four additional washes were performed with ultrapure
132 water, following the same centrifugation parameters. Finally, the formulations were collected for further
133 experimentation.

134 Loading of LY2157299. LY2157299, previously dissolved in DMSO, was added to the lipids dissolved in 100
135 µL of 96% EtOH, at a theoretical loading of 10% (w/w with respect to the total lipid content), prior injection
136 in the aqueous phase. VitE:SM nanoemulsions loaded with LY2157299 (VitE:SM:LY) were obtained. HPLC
137 analyses, performed in an HPLC system 1260 Infinity II (Agilent, USA) equipped with a pump G7111A, an
138 autosampler G7129A, and UV-Vis detector G7114A set at 236nm, and with a LUNA C18 column (5µm, 100Å
139 pore size, 4.6 mm × 150 mm) (Phenomenex Inc., US) operating at 25 °C, was used for quantification of the
140 encapsulated drug. The mobile phases used were solvent A (0.1% acetic acid, constant), solvent B (50%
141 methanol), and solvent C (50% acetonitrile). The flow rate was set at 1 mL/min. Separation was achieved by
142 mobile phase; drug peak (LY) at minute 2, VitE/SM peak at minutes 11-12. Total run 14 minutes.
143

143 **Preparation of TF labelled VitE:SM nanoemulsions.** Fluorescent labelled VitE:SM nanoemulsions were
144 formulated as previously described in the sections “Lipid nanosystems preparation” and “Loading of
145 LY2157299”. Briefly, sphingomyelin-TopFluor® (4.5 µg) (Cat No. 810265, Avanti Polar Lipids) and/or
146 LY2157299 (0.55 mg) (Cat No. HY-13226, MedChem) were combined with other organic phase compounds
147 (VitE and SM) in a final volume of 100 µL of 100% EtOH. The resulting organic phase was swiftly injected into
148 1 mL of ultrapure water, immediately forming the nanosystems at RT.
149

149 **Physicochemical characterization of the lipid nanosystems.** Particle size and polydispersity index (PDI)
150 were determined by Dynamic Light Scattering (DLS) and Z-potential values by Laser Doppler Anemometry
151 (LDA), using a Zetasizer NanoZS® (Malvern Instruments, Worcestershire, UK). Samples were diluted 1:10 with
152 ultrapure water and the measurements were performed at 25°C with a detection angle of 173°.
Subsequently, all developed formulations were evaluated in relevant biological media to ensure the maintenance of their properties during *in vitro* assays. Formulations were incubated at 37°C with 1% FBS (Fetal Bovine Serum) supplemented RPMI 1640 (Corning) at a 1:6 (v/v) dilution under orbital shaking (300 rpm). Additionally, VitE:SM mean particle size and morphology distribution were observed by Field Emission Scanning Electron Microscopy (FESEM) using a ZEISS FESEM ULTRA Plus microscope (Carl Zeiss Micro Imaging, GmbH, Germany) configured with InLens and STEM modes and operating at 20 kV. Twenty µL (0.50 mg/mL) of filtered nanosystems were stained with phosphotungstic acid (2% w/v) at a ratio 1:1. Next, 10 µL were placed on a carbon coated grid and left for 30s. The grid was allowed to dry and then, repeatedly washed with filtered ultrapure water. Finally, it was left dry until its visualization.

**Cell culture and reagents.** All murine (mu) and human (hu) cells utilized in the study (muBMDM, muBMDM, muKPC, huPANC1, huCAFs, and hu macrophages) were cultured in RPMI 1640 1X (Cat no. 15-040-CV, Corning) supplemented with 10% FBS (Gibco), 50 units/ml penicillin/streptomycin (Cat no. 15-040-CV, Gibco), L-Glutamine (Cat no. BE17-605E, Lonza) and Amphotericin B (Cat no. 15290-018, Gibco) at 37°C with 5% CO₂. IBMDM cells were obtained from Dr. Antonio Castrillo's laboratory (IIBM), and hu macrophages were obtained from excess buffy coat-derived PBMCs from Dr. Lisardo Boscá's laboratory (IIBM). Mu BMDM cells were extracted from the bone marrow of the femur and tibia of euthanized C57BL/6 mice and cultured with 10 ng/mL of murine M-CSF (Cat no. 315-02, PeproTech). The mu PDAC KPC cell line was derived from a spontaneous tumor originating in a genetically engineered LSL-Kras<sup>G12D/+</sup>; LSL-Trp53<sup>R172H/+</sup>; Pdx-1-Cre (KPC) C57BL/6 mouse model. Primary hu CAFs were established via an outgrowth method from a freshly digested primary human resectable PDAC tumor as already described⁵⁰. When fluorescence-marked cells were required, KPC cells and CAFs were infected with an mCherry expressing lentivirus (PRRL CMV IRES mCherry) as described in⁵¹, and PANC1 cells were fluorescently-labeled with a GFP expressing lentivirus (PRRL CMV GFP). Regular mycoplasma testing was conducted on all cells. For macrophage polarization, primary muBMDM or IBMDM cells were cultured for 24h with 10 ng/ml of IL-4 (Cat no. 214-14, PeproTech) for M2 or 10 ng/ml of LPS (Cat. no L4391, Sigma) + IFN-γ (Cat. no 315-05, Peprotech) for M1. IBMDM cells were seeded at a density of 4x10⁵ cells/p60 prior to Western blot and qRT-PCR analyses.
179 **Cellular toxicity assay.** The toxicity of nanoemulsions on macrophages was assessed using the Toxilight
180 BioAssay kit (Lonza, Walkersville, MD) in accordance with the manufacturer's instructions.

181 **RNA preparation and real-time PCR.** RNA from macrophage cultures and tissues was extracted using the
182 GTC method following the protocol described in⁵². For cDNA synthesis, 1 µg of isolated RNA was utilized in
183 conjunction with the Thermo Scientific Maxima First Strand cDNA Synthesis Kit (Cat. no K1671, ThermoFisher
184 Scientific), following the manufacturer's instructions. SYBR green-based qRT-PCR (Cat. no A25742,
185 PowerUp™ SYBR™ Green Master Mix, ThermoFisher Scientific) was performed using an Applied Biosystems
186 StepOnePlus™ real-time thermocycler (ThermoFisher Scientific). The PCR conditions included an initial 10-
187 minute denaturation step at 95°C, followed by 40 cycles of denaturation (15 sec at 95°C) and
188 annealing/extension (1 min at 60°C). mRNA copy numbers were determined relative to standard curves
189 composed of serial dilutions of plasmids containing the target coding sequences and normalized to *Actb*
190 levels. The primers used in this study are listed in Table S2.

191 **RNA sequencing analysis.** RNA was extracted from BMDM macrophages polarized to M2 with IL4 and
192 treated or untreated with VitE:SM nanoemulsions (1 mg/mL), following the procedures outlined in the
193 previous methods section. Genes with a fold change >1 and a p-value<0.05 were considered differentially
194 upregulated and genes with a fold change <-1 and a p-value<0.05 were considered differentially
195 downregulated after the comparison of gene expression in both M2 and VitE:SM samples. Genes were
196 ranked according to LOG2 fold change and Gene Set Enrichment Analysis was performed employing GSEA
197 v4.3.2. (Broad Institute). Gene signatures were obtained from Molecular Signature Database (MsigDB) and
198 from signatures published by other groups. Signatures with a NOM p-value<0.05 and an FDR q-value<0.05
199 were considered significantly enriched. Volcano plot and GSEA histogram were made employing Graph Pad
200 Prism 8 and GSEA plots were obtained with GSEA v4.3.2. FASTQ data files generated have been deposited in
201 Sequence Read Archive (SRA) of the National Center for Biotechnology Information (NCBI) with accession
202 number ID: PRJNA1099468.
**Western blot.** Cells were lysed in RIPA Buffer (Cat. no R0278-50mL, Sigma-Aldrich), supplemented with a protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN). The extracts were clarified for 15 minutes at 13,300 rpm at 4°C, and protein concentrations were determined using the BCA Protein Assay Kit (Cat no. 23227, Pierce). For Western blot analysis, 60 µg of clarified protein was separated on 10% SDS-PAGE gels and transferred to 0.45 µM PVDF membranes (Cat no. IPVH00010, Merck Millipore). For protein denaturalization, NuPAGE (Cat no. NP0007, Invitrogen) and 2-Mercaptoethanol (Cat no. M7522, Sigma) were used. Membranes were blocked with PBS-T (PBS 1X + 1% Tween20 (v/v) (Cat no. BP337-5000, Fisher bioreagents)) + 5% BSA (w/v) (Cat no. MD04602, Nzytech) and incubated with anti-mouse ARG1 (1:1000, Cat no. sc-271430, Santa Cruz Biotechnology) or anti-mouse beta Tubulin (1:2000, Cat no. E-AB-20033, Elabscience) overnight at 4°C. Three washes with PBS-T were performed before and after incubation with secondary horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse antibodies (Amersham) for 1 hour. Western blots were developed using SuperSignal chemiluminescent substrate (Cat no. 34577, Thermo Scientific), and images were captured using an iBright™ 1500 Imaging System (Invitrogen). Densitometric analysis was conducted using ImageJ, and indicated molecular weights were obtained from a PageRuler™ Plus Prestained protein ladder (Cat no. 26619, Thermo Scientific).

**FFPE embedded tissue dissociation.** Tissues embedded in paraffin were sectioned at 50 microns using a microtome, and a single-cell suspension was prepared with the MACS FFPE Tissue Dissociation Kit (Miltenyi, Cat. No 130-118-052). Briefly, tissue sections were deparaffinized, then dissociated chemically, enzymatically, and mechanically, followed by washing and filtration. After obtaining the single-cell suspension, the cells were blocked and stained for flow cytometry.

**Flow cytometry.** Mouse cell cultures or cells extracted from tissues were blocked with purified anti-mouse CD16/CD32 (Fc Shield) (1:100, Cat no. 70-016-U500, TONBO) for 10 min at 4°C and then, they were washed with 1X PBS prior to cell surface marker incubation for 30 min at 4°C with the following antibodies: CD45 (PE-Cyanine7), CD11b (PerCP-Cyanine5.5), F4/80 (APC) and CD206 (PE) or CD45 (PE-Cyanine7)/CD206 (PE-Cyanine7), CD11b (PerCP-Cyanine5.5) and F4/80 (APC) for the intrasplenic experiment (Table S3). Human
macrophages were blocked with Flebogamma (Grifols) for 15 min at 4°C and then resuspended in Flow buffer [1X PBS; 3% FBS (v/v); 3mM EDTA (v/v)] before analysis. For detection of M2 macrophage markers on control and nanoemulsion-treated human M2-polarzied macrophage cultures, the mouse monoclonal anti-human-CD163-PE antibody and the mouse monoclonal anti-human-CD206-FITC antibody were used (Table S3). Prior to analysis, all samples were washed with 1X PBS and cells were analyzed with a 4-laser Attune Nxt Acoustic Cytometer (Thermo Fisher Scientific). To exclude dead cells, samples were resuspended in Flow buffer [1X PBS; 3mM EDTA (v/v)] with 2µg/mL of DAPI before analysis. Results were analyzed with FlowJo software (Tree Star Inc., Ashland, OR.).

**Microscopy images.** For nanoparticle visualization, BMDM cells were seeded in Glass Bottom Dish 35 mm plates (Cat no. P35G-1.5-14-C, Mattek) and treated for 24h with VitE:SM:TF nanoemulsions (0.5mg/mL). Cells were washed with 1X PBS prior to Hoechst 33342 (Cat no. 62249) staining of nuclei. Images were acquired with a laser scanning confocal microscope Zeiss 710 40X Apochromatic and were analyzed using the software ZEN 2009 and ImageJ. Culture plate images were acquired with an Invitrogen™ EVOS™ Digital Color Fluorescence Microscope (Cat no. 12-563-340, Thermo Fisher Scientific).

**Tissue digestion and cell isolation.** Tissues extracted from mice were minced and enzymatically digested with Collagenase P (Cat no. 11213857001, Roche) and Dispase (Cat no. 17105-041, Gibco) for 30 min at 37°C, vortexing every 10 minutes. Samples were passed through a 40µM Fisherbrand™ Sterile Cell Strainer (Cat no. 11587522, FisherScientific) and cells were collected in a new tube, where they were centrifugated at 1,300 rpm for 5 min. After a 1X PBS wash, cells were ready for flow cytometric analysis as described above.

**Histology.** For histopathological analysis, a section of each organ was fixed in 10% buffered formalin and paraffin embedded. Formalin-fixed paraffin-embedded (FFPE) blocks were serially sectioned (3 µm thick) with a microtome LEICA RM 2125 RTS and stained with hematoxylin and eosin (H&E). The stained sections were covered with DPX mounting medium fast (Cat no. 255254.1610, Panreac AppliChem) and a coverslip. Images were acquired with a Motic EasyScan One slide scanner and analyzed with Motic DS Assistant.
**DNA extraction and PCR.** DNA from tissue were extracted with QIAcube Connect equipment (Cat no. 9002864, QIAGEN), TissueLyser LT (Cat no. 85600, QIAGEN) and DNeasy Blood & Tissue kit (Cat no. 51106, QIAGEN) following the manufacturer's instructions. PCR amplification was performed with DreamTaq Green PCR Master Mix (2X) (Cat no. K1081, Thermo Scientific) and a SimpliAmp™ (Cat no. A24812, ThermoFisher Scientific) thermocycler. The amplification protocol was as follows: 5 min denaturation step at 95°C, 35 cycles of denaturation (30 sec at 95°C), annealing (30 sec at 67°C) and extension (45 sec at 72°C), and a final extension of 5 min at 72°C. PCR products were resolved in an agarose (1%) gel (Cat no. 8019.00, Condlab) with SybrSafe (Cat no. S33102, Invitrogen) staining for 45 min at 100V, and images were acquired with an iBright™ 1500 Imaging System (Invitrogen). As a reference for molecular weight, the 1kb Plus DNA Ladder (Cat no. 10787018, Invitrogen) was used. Primers used for CRE and murine *Gapdh* amplification have the following sequences: CRE: forward: 5'-gcctgcattaccggtcgatgcaacga-3'; reverse: 5'-gtggcagatggcgcggcaacaccatt-3', GAPDH forward: 5'-gcctgcattaccggtcgatgcaacga-3'; reverse: 5'-gtggcagatggcgcggcaacaccatt-3'.

**Magnetic resonance imaging (MRI).** Anesthetized animals (induced with 2-2.5% and maintained with 1-1.5% isoflurane/oxygen) were placed in a supine position in a methyl methacrylate holder in the MRI system, and restrained with a bite-bar attached to a nose mask and a small piece of tape over the head and attached to the animal's bedding. A heated probe was employed to maintain the core body temperature at approximately 37°C. Physiological status of the animals was monitored by a gating system designed for small animals using respiratory rate and body temperature. Image acquisitions were carried out in a Bruker BioSpec® 7 T system (Bruker Biospin, Ettlingen, DE), 16 cm bore, with a 90 mm gradient insert of 360 mT/m and a 40 mm quadrature volume resonator, interfaced with an Avance III radiofrequency console and running ParaVision 6.0.1 software. Anatomical T2-weighted images were acquired in axial coronal orientation for tumor assessment: TR (repetition time): 1200 ms; TE (echo time): 30.0 ms; FA (flip angle): 180°; FoV (field of view): 40 mm × 40 mm; matrix: 264 × 264; 24 slices of 0.8 mm per slice, acquisition time: 15 min per orientation.
277 **In vivo orthotopic experiments.** Mice were housed according to previously reported protocols⁵³. All *in vivo* procedures in mice were conducted in accordance with protocols approved by the local Animal Experimental Ethics Committee of the Instituto de Salud Carlos III (PA 34-2012) or the Use Committee for Animal Care from the Universidad Autónoma de Madrid (UAM) (Ref# CEI-25-587) and the Comunidad de Madrid (PROEX 335/14 or 294/19). C57BL/6 mice (Charles River Co), aged 6-9 weeks, underwent orthotopic pancreatic implantation with KPC cells (2,500 cells/pancreas) resuspended in 50 µL of a 3:2 ratio of Matrigel (Cat no. 354234, Corning):RPMI. Treatments commenced 4-6 days post-implantation, with nanoemulsion doses of 69 mg/kg administered intraperitoneally and 25 mg/kg administered retro-orbitally. For nanoemulsion concentration or buffer exchange, Centrifugal Filters Amicon Ultra - 0.5mL Ultracel-100K (Cat no. UFC510096, Merck Millipore) were employed following the manufacturer's instructions. Three weeks after treatment initiation, mice were euthanized and tumors and livers were weighed and photographed after animal perfusion with cold 1X PBS.

289 **Liver function test.** Blood samples were taken by cardiac puncture. EDTA tubes were centrifuged and plasma was collected. Determination of liver enzymes were performed by a clinic certificated laboratory (Laboratorios Echevarne, Spain) and normal values references were provided by Charles River Co https://www.criver.com/sites/default/files/resources/doc_a/C57BL6MouseClinicalPathologyData.pdf.

293 **Intrasplenic and intravenous model.** C57BL/6 mice (Charles River Co), aged 6-9 weeks, underwent intrasplenic implantation of KPC/mCherry cells (25,000 cells/spleen) resuspended in 1X PBS following a previously reported protocol⁵⁴. Treatments started 2 days post-implantation, with nanoemulsion doses of 25 mg/kg administered retro-orbitally. Three weeks post-treatment initiation, mice were euthanized and perfused with cold 1X PBS; subsequently, the weights of the tumors and livers were measured and documented through photography. For the intravenous model, KPC cells were suspended in 1X PBS and then injected into the tail vein of C57BL/6 mice at a concentration of 25,000 cells per injection.

300 **Peripheral blood mononuclear cells extraction.** Excess PBMCs were distributed across three 6-well culture plates per donor, utilizing RPMI 1640 complete medium. After 24 hours, suspended lymphocytes
302 were washed with 1X PBS and adherent human monocytes were prepared for following experiments. For 303 human macrophage differentiation we employed 60 ng/mL of MCSF (PeproTech) to polarize them to M2 304 macrophages as previously described⁵⁵.

305 **Spheroid formation and orthotopic implant.** For the 3D spheroid model³⁶, suspensions of PANC1/GFP 306 (1,000 cells/well) and CAFs/mCherry (4,000 cells/well) cells were prepared in DMEM complete medium 307 supplemented with 25ng/mL of human fibroblast growth factor (FGFb) (Cat no. GF446-100G, Sigma). The 308 resulting mixture was transferred into each well of 96-well ultra-low attachment (ULA) plates. After cell 309 seeding, the plates were centrifuged (200×g, 5 min, RT) and then placed in a humidified atmosphere with 5% 310 CO₂ at 37°C. On day 3, monocytes from PBMCs (4,000 cells/well) were added, and spheroid treatment with 311 nanoemulsions (1 mg/mL) was carried out from day 4 to day 7, after which fresh medium was added. On day 312 10, NOD/ShiLtSz-Prkdcscid (NOD.SCID) mice (Charles River Co) aged 15-20 weeks were orthotopically 313 implanted in the pancreas with 40-50 disaggregated spheroids, which were trypsinized, centrifuged, and 314 resuspended in 50 µL of a 3:2 proportion of Matrigel (Cat no. 354234, Corning):RPMI. Three months after 315 implantation, mice were euthanized to evaluate tumor growth and metastasis using various techniques after 316 animal perfusion with cold 1X PBS.

317 **Statistical analyses.** Results are presented as means ± standard deviation (SD). To compare more than two 318 sets of data one-way ANOVA with Dunnett's multiple comparisons statistical analysis was performed while 319 an unpaired t-student tests was used to analyzed two data sets. All of the statistical analyses were 320 performed with Graph Pad Prism 8. A statistically significant difference was considered as *p < 0.05; **p 321 <0.01; ***p < 0.001; and ****p < 0.0001, as indicated in the figure legends.

322 **RESULTS AND DISCUSSION**

323 **Development and characterization of lipid nanosystems.** In this study, VitE:SM nanosystems are 324 proposed for targeting TAMs in the liver to treat pancreatic cancer metastasis. As stated in the introduction, 325 these type of nanocarriers have shown a high potential for the delivery of therapeutics to treat cancer³⁴ and
proved that they can penetrate dense cancer spheroids, specifically in pancreatic cancer³⁶,⁵⁶. The formulated compositions were prepared by adapting the ethanol injection method, enabling a single-step preparation and the straightforward formation of the nanosystems³⁴, ³⁵, ³⁷ (Figure 1a). Sphingomyelin (SM) is composed of two long carbon chains (hydrophobic tail), a secondary amine group acting as a linker and a phosphate group (hydrophilic head). Considering its lipophilicity, it is expected that it can form emulsions when combined with oils such as Vitamin E (VitE), a fact that has been proved experimentally and through computational simulation studies, selecting a mass ratio VitE:SM 1:0.1 as suitable for the association of therapeutics³⁴.

Alternative oils, can also form nanoemulsions, in combination with SM, following the same rational and experimental approach. The nanoemulsion composition could also be complemented by additional phospholipids, including Phosphatidylcholine (PC), Phosphatidylinositol (PI), Phosphatidylserine (PS), Phosphatidylglycerol (PG), or Palmitic acid (PA), since there is growing evidence suggesting that certain phospholipids can influence TAM reprogramming⁵⁷,⁵⁸. Consequently, the hydrophobic carbon chain can be incorporated into the oily core of the structure, while the hydrophilic phosphate head is exposed on its surface.

In this study, VitE:SM nanoemulsions have been proposed for targeting and reprogramming TAMs in the liver, considering the high potential of this formulation in the development of cancer therapeutics, its preferential biodistribution to the liver, and the role of VitE and SM in modulating inflammation, as previously described⁴⁵,⁴⁶. Alternative compositions have been formulated for comparative purposes, and are detailed (Figure 1b, Table 1). Following formation, all formulations showed appropriate physicochemical properties, with minor differences in size and surface properties, rendering homogeneous populations. In terms of colloidal stability, the formulations were stored at 4°C, and their physicochemical properties, such as particle size, population homogeneity (PDI) and zeta potential were monitored over time. Formulations containing VitE, either with SM or PC (1:0.1 w/w), remained stable for at least 30 days (Figure S1a-b), in agreement with previous results showing high stabilities for VitE:SM nanoemulsions³⁴.

Subsequently, the lipid nanosystems were incubated with complete RPMI cell culture medium supplemented with 1% FBS at 1:6 (v/v) and measured for up to 24h at 37°C under orbital shaking. Their size
and PDI were determined at different time points by further diluting the sample in ultrapure water at 1:10 (v/v). In line with the results, all formulations showed a good stability with minimal increase in particle size and polydispersity over time (Figure S1c-k).

**The lipid nanosystems comprising VitE:SM demonstrate the most favorable M2 macrophage reprogramming effects.** Since our primary objective was to reprogram macrophages with nanotechnology, we first assessed the behavior of VitE:SM nanoemulsions in relation to nanosystems with different lipid compositions (Figure 1b, Table 1) in immortalized murine bone-marrow-derived macrophages (IBMDM)⁵⁹ to determine their potential toxicity and impact on M2 macrophage reprogramming.

<table><thead><tr><th>Formulation</th><th>Mass ratio</th><th>Size (nm)</th><th>PDI</th><th>ZP (mV)</th></tr></thead><tbody><tr><td>VitE:SM</td><td>1:0.1</td><td>97 ± 5</td><td>0.08 ± 0.02</td><td>-13 ± 8</td></tr><tr><td>VitE:OA:SM</td><td>0.5:0.1:0.5</td><td>101 ± 3</td><td>0.21 ± 0.02</td><td>-27 ± 1</td></tr><tr><td>M:SM</td><td>1:0.1</td><td>93 ± 6</td><td>0.10 ± 0.05</td><td>-10 ± 4</td></tr><tr><td>VitE:PC</td><td>1:0.1</td><td>125 ± 6</td><td>0.04 ± 0.01</td><td>-5 ± 2</td></tr><tr><td>VitE:SM:PI</td><td>1:0.1:0.002</td><td>66 ± 3</td><td>0.20 ± 0.06</td><td>-4 ± 2</td></tr><tr><td>VitE:SM:PS</td><td>1:0.1:0.002</td><td>91 ± 7</td><td>0.22 ± 0.04</td><td>-7 ± 2</td></tr><tr><td>VitE:SM:PG</td><td>1:0.1:0.002</td><td>97 ± 4</td><td>0.10 ± 0.02</td><td>-12 ± 3</td></tr><tr><td>VitE:SM:PA</td><td>1:0.1:0.05</td><td>100 ± 8</td><td>0.19 ± 0.03</td><td>-12 ± 1</td></tr><tr><td>VitE:SM:PA002</td><td>1:0.1:0.002</td><td>95 ± 2</td><td>0.09 ± 0.02</td><td>-10 ± 4</td></tr></tbody></table>

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**Table 1.** Physicochemical properties of lipid nanosystems measured, after preparation, by dynamic light scattering (DLS) and laser doppler anemometry (LDA) including hydrodynamic size, polydispersity index (PDI) and zeta potential (ZP). Results are expressed as mean ± SD, n = 5. Abbreviations: nm: nanometer, PDI: polydispersity index, ZP: zeta potential in millivolts (mV).

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366 IBMDM macrophages were polarized for 24 hours with IL-4 to induce M2 polarization and with LPS+IFN-γ for 367 M1 polarization. Following this, the M2 macrophages were treated with nanoemulsions for 4 hours to assess 368 toxicity and reprogramming by qPCR analysis after a 24-hour post-treatment period, and by Western 369 blotting to evaluate the expression of the principal M2 marker Arg1 at 48 hours post-treatment (Figure 2a). 370 To assess associated toxicity induced by the different formulations, a bioluminescent assay that measures 371 the release of adenylate kinase from damaged cells was employed, revealing that VitE:SM was one of the 372 least cytotoxic formulations (Figure 2b), which was confirmed by light microscopy (Figure S2a). VitE:OA:SM 373 was discarded due the high cytotoxicity observed (Figure S2a).

374 Regarding activation, M2 macrophages were treated with nanoemulsions (Figure 2a) and by qPCR and 375 Western Blot their reprogramming towards a more M0/M1 phenotype was assessed by analysis of Arginase 376 1 (Arg1/ARG1) expression, a well-known marker for M2 macrophages in PDAC⁶⁰. By qPCR analysis we 377 observed a general decrease in Arg1 expression with all the tested formulations, with a more pronounced 378 decrease observed in the case of the VitE:SM formulation compared to untreated M2 macrophages (Figure 379 2c). This trend was similarly observed at the protein level. While a decrease in ARG1 expression was 380 achieved with nearly all the formulations, it was only statistically significant in the case of nanoemulsions 381 containing VitE:SM and VitE:SM:PI (Figure 2d).

382 Based on these data, we determined that the most effective formulation for macrophage reprogramming 383 and the most suitable composition for further development was the nanosystem containing VitE:SM (Figure 384 2e). To investigate the potential synergies offered by VitE and SM in TAM reprogramming, we conducted a 385 new experiment in which IBMDM macrophages were first polarized with IL-4 for 24 hours to induce M2 386 polarization. The M2 macrophages were then treated for 4 hours with nanoemulsions (VitE:SM), SM, VitE, 387 and a free combination of VitE and SM to assess toxicity (% live cells) and M2 reprogramming via flow 388 cytometry (CD45<+CD11b+<F480+<CD206+) 24 hours post-treatment (Figure S2b). Our results confirm that 389 the VitE:SM composition of the nanoparticle exhibits a synergistic effect in reprogramming M2 TAMs and 390 demonstrates improved cell viability compared to the other compositions. To further analyze the structure 391 of this composition, we employed field emission scanning electron microscopy (Figure S3).
We then investigated whether primary murine macrophages derived from bone marrow monocytes (BMDM) treated with VitE:SM maintained their reprogramming over time and whether these effects were reversible upon subsequent exposure to the M2-polarizing cytokine IL-4 (Figure S4a). Indeed, Western blot analysis of ARG1 revealed a continued reduction of this M2 marker over time in BMDMs treated with the nanosystem. Additionally, VitE:SM nanoemulsion-treated macrophages that were repolarized with IL-4 did not increase ARG1 to levels observed in control groups (Figure S4b). Considering the implications for in vivo use, these findings suggest that despite the continuous influence from tumor secreted pro-TAM factors, treated macrophages are likely to retain the polarization state induced by the VitE:SM nanoemulsions over time.

We hypothesized that the potent reprogramming capacity of the VitE:SM formulation arises from two key properties. On the one hand, it is well-established that the phagocytosis of lipid membranes by macrophages affects their programming and metabolism³², ⁶¹, ⁶². Some studies utilize cell membrane-derived nanoparticles (nanoghosts) for macrophage reprogramming, attributing this effect to components like cytokines and chemokines present in the cell membranes of nanoghosts. However, it is also plausible that membrane sphingolipids such as SM play a crucial role in macrophage reprogramming⁴³⁻⁴⁶, ⁶². On the other hand, the immunostimulant properties of VitE may also contribute to macrophage reprogramming³⁹⁻⁴¹. Thus, the dual actions of VitE and SM suggests a potential synergy in enhancing the reprogramming capacity of this composition, making this combination particularly promising for further experimentation.

**RNAseq analysis of M2 and M2 VitE:SM nanoemulsion-treated macrophages.** To further investigate the effects of the VitE:SM formulation on M2 macrophage polarization at the transcriptomic level, we performed RNAseq on IL-4-stimulated (M2) and IL-4-stimulated VitE:SM nanoemulsion-treated BMDMs. Among the genes significantly upregulated in VitE:SM-treated macrophages, we found genes related to inflammation (i.e., *Tnf*, *Ptgs2*, *Il-11*, *Il-1a*, *Cxcl1*) and with antigen presentation (i.e., *H2-M2*). Additionally, among the downregulated genes, we identified *Cd51* and *Cd300e*, which are related to M2 polarization, and *Angpt1* which is a pro-angiogenic factor (Figure 3a).
We next performed a Gene Set Enrichment Analysis (GSEA) employing Molecular Signature Database (MsigDB) signatures (including Gene Ontology, Reactome and Hallmarks) or custom gene signatures. Interestingly, VitE:SM nanoemulsion-treated macrophages recover a pattern related to resting differentiated macrophages or monocytes, rather than a gene set associated with M2 or IL-4 stimulated macrophages. We also found mainly an enrichment in gene signatures related to TNF-TNFR1 signaling, but also macrophage activation via Toll receptors, interferon-related signaling, reactive oxygen species and signatures related to innate immune system activation, indicating that VitE:SM treatment induces a reprogramming of M2 macrophages towards a more inflammatory and less immunosuppressive phenotype, which is in line with our previous results and hypothesis (Figure 3b-c).

**The intraperitoneal injection of VitE:SM nanoemulsions reduces tumor burden in an orthotopic KPC tumor model.** To initially validate the *in vivo* efficacy of the nanosystems, we conducted a preliminary study employing murine PDAC cells obtained from spontaneous tumors from the KPC (LSL-Kras<sup>G12D/+</sup>; LSL-Trp53<sup>R172H/+</sup>; Pdx-1-Cre) mouse model<sup>63</sup>. These cells were orthotopically injected into the pancreas of syngeneic and immunocompetent C57BL/6 wild-type (WT) mice (Figure 4a), with the nanosystem treatment commencing on the seventh day post-implantation in two distinct non-parallel experiments. In each experiment, randomized groups of 4 or 5 mice were intraperitoneally injected with vehicle solution (0.9% NaCl) for the control mice or 69 mg/kg of VitE:SM nanoemulsions for treated mice. This regimen was applied for five consecutive days per week. Mice were euthanized on day 27 post-implantation, after confirming tumor growth in a sentinel mouse by Magnetic Resonance Imaging (MRI) (Figure S5a). Total body weight was recorded at the start and end of the treatment to assess nanoemulsion toxicity, revealing no differences between groups throughout the study period (Figure 4b). In this model, tumor presence typically impairs weight gain and often leads to tumor-associated cachexia, so the lack of weight gain is expected. Full-body necropsy was then performed and no liver toxicity was observed at the organotypic level (Figure 4c), nor were differences observed in liver weight (Figure 4d). At the histopathological level, examination of H&E-stained liver slides show that the structure and architecture of the liver remain unaffected by the treatment. There is no evidence of inflammation, necrosis, or loss of architecture in the central vein or sinusoids. (Figure
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4e). Finally, the assessment of liver toxicity following treatment with VitE:SM nanoemulsions, as determined by liver function tests, did not reveal elevated values of ALT, AST and GGT when compared to standard reference ranges (Table S3). Liver toxicity was also absent in non-implanted mice treated with three doses of VitE:SM nanoparticles administered every two days over a one-week period. Serum analysis after treatment showed normal ALT enzyme levels, with no significant difference between control and VitE:SM mice (Figure S5b). Additionally, the mice did not experience weight loss during the treatment process (Figure S5c). This underscores the biocompatibility of the chosen nanosystem, as evidenced by the absence of toxicity associated with the nanoemulsion. Moreover, unlike other treatments that deplete TAMs, such as clodronate or toxin-conjugated monoclonal antibodies³¹, ⁶⁴, VitE:SM nanosystems could offer a safer therapeutic option.

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Both at the level of the tumor alone (Figure S5d) and including the adjacent peritoneal tumor-derived masses (Figure 4f-g), a reduction in total tumor size was evident in mice treated with the VitE:SM nanosystem compared to the control group. (Figure 4f-g and S6). An additional control using non-effective nanoparticles M:SM in M2 TAM reprogramming was included to specifically highlight the potential of the VitE:SM composition. Tumor weights confirmed a significant decrease in tumor burden with intraperitoneal administration of VitE:SM nanoemulsions, an effect not observed with M:SM nanoparticles (Figure 4g). These findings align with other studies that have also utilized nanoparticles to stimulate the immune system. The integration of nanotechnology into biomedicine has demonstrated a consistent trend in enhancing immune responses, highlighting the efficacy of nanoparticle-based approaches in immunomodulation⁶⁵, ⁶⁶.

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**Targeting capacity of the TopFluor® (TF) labelled VitE:SM nanoemulsions in vitro and in vivo.** To further evaluate the *in vivo* application of the nanosystems, nanoemulsions were formulated using sphingomyelin labeled with the green fluorophore TopFluor® (TF) for tracking purposes in mice (Figure 5a and Table 2). First, confocal microscopy images of IBMDM macrophages revealed green fluorescence in those cultures treated with VitE:SM:TF nanoemulsions (Figure 5b). Detection of the fluorophore by flow cytometry, using Ex488nm Em530/30nm, showed nearly complete staining of macrophages (Figure 5c).

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Since the goal of this study was to target liver TAMs, we assessed which route of administration would favor liver biodistribution, as measured by flow cytometric analysis of tumors, liver and lungs. VitE:SM:TF nanoemulsions were administered intraperitoneally or retro-orbitally to KPC orthotopically implanted C57BL/6 mice seven days post-surgery, at a dose of 25 mg/kg of VitE:SM:TF nanoemulsions. Mice were euthanized 48 hours later to assess TF biodistribution in the tumor and the two principal organs susceptible to develop metastasis: liver and lungs. Following organ digestion, we confirmed by flow cytometry that while intraperitoneal administration is optimal for reaching the tumor, retro-orbital injection is more effective in reaching the liver and the lungs (Figure 5d). To further compare retro-orbital injection with another intravenous route, such as tail vein injection, we conducted an additional biodistribution experiment, which confirmed that retro-orbital injection was more efficient in delivering the treatment to the liver, the primary target organ of this study (Figure 5d). To determine the kinetics of VitE:SM:TF nanoemulsion liver targeting, and specifically liver macrophage (i.e., CD45+, CD11b+, and F4/80+) targeting (Figure 5e), VitE:SM:TF fluorescence was assessed 1h, 4h, 24h, and 72h post-injection. Flow cytometry analysis of digested livers indicated that peak accumulation of VitE:SM:TF in liver macrophages was reached at 24h, with a decrease at 72h (Figure 5f). Thus, we established that the optimal treatment regimen for subsequent *in vivo* intervention studies to be retro-orbital injection every 48h.

<table><thead><tr><th>Formulation</th><th>Mass ratio</th><th>Size (nm)</th><th>PDI</th><th>ZP (mV)</th><th>%EE LY</th></tr></thead><tbody><tr><td>VitE:SM</td><td>1:0.1</td><td>97 ± 5</td><td>0.08 ± 0.02</td><td>-13 ± 8</td><td>-</td></tr><tr><td>VitE:SM:TF</td><td>1:0.1:0.001</td><td>102 ± 4</td><td>0.07 ± 0.03</td><td>-6 ± 1</td><td>-</td></tr><tr><td>VitE:SM:LY</td><td>1:0.1:0.1</td><td>107 ± 5</td><td>0.09 ± 0.02</td><td>-12 ± 2</td><td>92 ± 2</td></tr></tbody></table>

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<caption>Table 2. Physicochemical properties of VitE:SM, VitE:SM:TF and VitE:SM:LY nanosystems measured, after preparation, by dynamic light scattering (DLS) and laser doppler anemometry (LDA) including hydrodynamic size, polydispersity index (PDI), zeta potential (ZP) and encapsulation efficiency (%EE) of LY. Results are expressed as mean ± SD, n = 5. Abbreviations: nm: nanometer, PDI: polydispersity index, ZP: zeta potential in millivolts (mV).</caption>
489 Loading of VitE:SM nanoemulsions with the TGF-βR1 inhibitor (LY2157299) reduces tumor growth and diminishes TAM liver infiltration. We decided to further explore the potential of VitE:SM nanoemulsions to encapsulate and deliver the TGF-βR1 inhibitor Galunisertib LY2157299, referred to as LY (VitE:SM:LY) (Figure 6a and Table 2). We hypothesized that LY would inhibit the activity of fibrotic cytokines *in vivo* and prevent the M2 polarization of macrophages via TGF-β<sup>67-69</sup>, as previously discussed, thereby further obstructing the formation of the pre-metastatic niche in the liver<sup>47-49</sup>. Moreover, the encapsulation of LY into the VitE:SM nanosystem should in theory enhance its delivery and mitigate non-specific toxicity often associated with the administration of free LY<sup>70, 71</sup>. We first tested the VitE:SM and VitE:SM:LY compositions at different concentrations (1 and 0.5 mg/mL) in BMDM cells polarized to an M2 state with IL-4. Western blot analysis of ARG1 protein levels revealed a decrease in this M2 marker across all treated samples, with no significant differences observed between different concentrations or with LY encapsulation (Figure 6b).
500 Additionally, qPCR analysis of various M1 or M2 markers consistently demonstrated a decrease in classical M2 markers such as *Arg1* and *Egr2* (Figure 6c) and an increase in some M1 markers such as *Cd86*, *Il1b*, and *Il12b* (Figure 6d). Of note, the addition of LY to the nanoemulsions did not lead to an improvement in macrophage reprogramming, which is in slight contrast to what we have previously seen with another TGF-βR1 inhibitor in human M2-polarized macrophages<sup>55</sup>. We speculated that the potent effect of the VitE:SM vehicle was masking the influence of LY *in vitro*, but we also reasoned that the additive effect offered by encapsulating LY in the nanosystems would be more evident *in vivo*.

507 Towards this end, C57BL/6 mice were orthotopically implanted with KPC cells, and VitE:SM:TF or VitE:SM:LY:TF nanoemulsion treatment was initiated on day 4 post-implantation at a dose of 25 mg/kg, administered retro-orbitally every 48 hours (3 days per week). Three euthanize time points to assess tumor progression were established: days 21, 28, and 35 post-implantations (Figure 7a). At the indicated time points, tumors were extracted, and a reduction in tumor size in mice treated with empty nanoemulsions (VitE:SM:TF) and with nanoemulsions loaded with LY (VitE:SM:LY:TF) was observed (Figure 7b). Monitoring tumor weight over time revealed that VitE:SM:TF and, more significantly, VitE:SM:LY:TF exerted a cytostatic effect compared to control mice (Figure 7c). Likewise, the sum of the data across all time points collectively
exhibited a significant reduction in tumor weight in mice treated with both nanoemulsion formulations (Figure 7c). Histological analysis of tumor samples revealed a higher proportion of tumoral area in mice from the control group, whereas areas of healthy pancreas were still prominent in many mouse pancreata from the treatment groups (Figure 7d-e).

Despite attempts to detect liver metastases in the orthotopic model, no macroscopic metastases were found at the experimental endpoint (d35) (Figure S7a) in any group, nor did our histological analyses of the liver reveal evident areas of tumor micrometastases (Figure S7b). Since our goal was to target liver macrophages in the hope of reprogramming TAMs, livers pieces were digested for flow cytometric analysis, revealing that not only did the nanosystems reach the macrophages in the liver (Figure S7c), but a reduction in the total population of CD45+/CD11b+/F4/80+ infiltrated macrophages with nanoemulsions treatments was observed (Figure 7f). Moreover, we analyzed the macrophage population characterized by CD206 expression, a parallel M2 marker for flow cytometry, and found a significant reduction in these TAMs with LY encapsulated nanoemulsions (Figure 7g), reinforcing our objective of M2 TAM targeting and reprogramming in the liver. While our data support macrophage reprogramming as the mechanism of action by which the VitE:SM nanoemulsions affect tumor growth and TAM liver infiltration, we cannot rule out a possible direct effect on tumor cells; although, flow cytometry analysis showed a preferential accumulation in macrophages versus the rest of live cells (Figure S7d).

To explore the immune profiling and metastasis-related cytokine response induced by nanoparticle treatment, we extracted RNA from liver and tumor tissues and performed qRT-PCR analysis on key cytokines. In both the liver (Figure S7e) and tumor (Figure S7f), treatment with VitE:SM, and especially VitE:SM:LY, led to a significant upregulation of pro-inflammatory and Th1-type cytokines (Il12, Tnf, Ptgs2, Cxcl1, Gzmb, and Il6), which are crucial for activating immune cells like macrophages and T cells. This pro-inflammatory shift was further supported by the downregulation of immunosuppressive cytokines (Arg1, Pglyrp1, Ccl2, and Mpo), contributing to a less permissive environment for metastasis. These findings align with previous studies on galunisertib, which also showed a shift towards a pro-inflammatory, anti-tumor immune response. Additionally, the *in vivo* results are consistent with *in vitro* data (Figures 2 and 3), where VitE:SM
and VitE:SM:LY effectively reprogrammed immunosuppressive M2 macrophages into a pro-inflammatory
M0/M1 phenotype, potentially reducing metastatic spread.

In contrast to the above detailed *in vitro* studies, this *in vivo* model highlights the synergistic impact of incorporating LY into the VitE:SM nanoemulsions. The results reveal superior outcomes with LY encapsulation compared to the effectiveness of VitE:SM nanosystems alone across all evaluated parameters, including tumor growth, the percentage of healthy pancreas in histological samples, and the reprogramming and infiltration of TAMs in the liver. This enhanced efficacy may be attributed to the physiologically relevant environment obtained in the *in vivo* setting where TGF-β signaling plays a crucial role in tumor progression and TAM recruitment to the liver<sup>16, 17</sup>. This underscored the significance of utilizing the LY-loaded VitE:SM nanoemulsions for our subsequent *in vivo* PDAC metastasis studies.

**TGF-βR1 inhibitor (LY2157299)-loaded VitE:SM nanoemulsions diminish liver metastasis in an**
**intrasplenic KPC metastasis model.** While the orthotopic model was an excellent and biologically-relevant model to study the effect of the nanoemulsions of primary tumor growth, we concluded that it was not the most suitable option for assessing liver metastasis, as the progression of the primary tumor was too rapid and precluded the development of macroscopic liver metastases within the time frame of the study (i.e., before humane endpoint of euthanasia). Consequently, we considered the feasibility of testing our treatment in a more appropriate metastasis model. In general, there are three models for studying PDAC metastasis: intravenous, intraperitoneal, and intrasplenic models, commonly utilized for investigating lung, peritoneal and lymph node, and liver metastases, respectively<sup>72</sup>. As our objective was to reprogram TAMs in the liver to prevent metastasis, the intrasplenic model<sup>54, 73</sup> represented a better option for our nanosystems, as injected cells have direct communication with the liver through the splenic vein (SV). However, a pilot experiment was conducted to compare intrasplenic versus intravenous injection of KPC cells in order to ascertain the most suitable model for validating our nanosystems. Consistent with findings in the literature<sup>72</sup>, PCR analysis revealed a higher number of CRE DNA copies (derived from KPC cells) in the liver
when using the intrasplenic model. Conversely, the intravenous model emerged as a more favorable option for promoting lung colonization (Figure S8).

KPC cells were injected into the spleens of C57BL/6 mice, after which cauterization and removal were performed to prevent the formation of primary tumors in the injected organ (Figure 8a). For these experiments, KPC/mCherry cells were used to track tumor cells in the different analyzed tissues. Retro-orbital administration of VitE:SM:LY:TF nanoemulsions (25 mg/kg) commenced on day 2 post-intrasplenic injection and was maintained 3 days per week until the experiment's endpoint on day 18 post-intrasplenic injection (Figure 8b). Upon euthanasia, we observed no differences in liver or tumor weight between the control and treated groups (Figure 8c); however, macroscopic liver metastases were detected only in the control mice (Figure 8d and Figure S9). Liver tissue is structured into distinct functional units known as lobules, featuring a hexagonal arrangement of portal triads surrounding a central vein and interconnected by sinusoids⁷⁴. In contrast to the treated mice, histological analysis of livers from control mice revealed loss of hepatocyte microarchitecture, including a reduction in the number of central veins and sinusoids, indicating tissue disorganization promoted by tumor progression (Figure 8e).

At the cellular level, flow cytometry analysis revealed a significantly lower proportion of KPC/mCherry cells in the livers of treated mice compared to controls (Figure 8f). Additionally, PCR analysis was performed to quantify CRE DNA copies (derived from KPC cells) in the liver, showing a reduction in amplification levels in the livers of treated mice compared to control mice (Figure 8g and S10a). Analyzing the CD206+ TAM population in the liver by flow cytometry (CD11b/F4/80+/CD206+), we observed, consistent with previous in vivo models, that the nanosystems reached liver macrophages (Figure S10b) and reduced the CD206+ TAM population (Figure 8h). To further investigate the immune profile following treatment, we prepared single-cell suspensions from representative paraffin-embedded livers for FACS analysis of different immune populations, including M1/M0 macrophages and T cells. After treatment, we observed again a reduction in CD206+ M2 macrophages, compensated by an increase in M0/M1 macrophages (Figure S10c). Additionally, there was an increase in the overall myeloid population and CD8+ T cells (Figure S10c), aligning with the
proinflammatory cytokine profile seen in the livers of treated mice (Figure S7e). Neutrophils, on the other hand, remained unchanged by the treatment (Figure S10c).

With this approximation, we successfully validated our hypothesis that reprogramming liver-infiltrating TAMs with the proposed lipid nanosystems has the potential to delay liver metastasis in PDAC. As previously mentioned, even in cases where the primary tumor is effectively resected, approximately 75% of patients experience metastatic relapse within five years post-surgical resection<sup>8-10</sup>. Thus, our therapeutic approach holds promise in preventing liver metastasis relapse for patients with resectable primary PDAC tumors, and could potentially be used in combination with chemotherapeutic interventions in patients with metastatic disease. The latter, however, still needs to be tested in experimental models.

**Ex vivo treatment of human 3D tumor/CAF/macrophage spheroids with VitE:SM nanoemulsions validates the translatability of the nanosystem therapy to humans.** Finally, we aimed to ascertain the translatability of our therapy to the human setting. To achieve this goal, we initially isolated and cultured monocyte-derived human macrophages. Subsequently, we induced M2 polarization using MCSF and then exposed the cells to different concentrations of VitE:SM nanoemulsions to evaluate their effect on M2 macrophage reprogramming. Using the immunosuppressive human TAM markers CD206 and CD163, we observed that VitE:SM nanoemulsions were also effective in reducing the M2 population *in vitro* (Figure S11a).

Building upon this preliminary result, we used an *ex vivo* 3D human spheroid cell model<sup>36</sup> to test the efficacy of our lipid nanoemulsions. In this scenario, empty VitE:SM nanoemulsions (without LY) were used since no differences in TAM reprogramming were observed between VitE:SM or VitE:SM:LY (LY-loaded nanoemulsions) *in vitro* (Figure 6b-d). Specifically, we generated *in vitro* spheroids containing human pancreatic cancer cells (PANC1), human CAFs, and human macrophages to recreate the PDAC tumor microenvironment in culture. These spheroids were treated with VitE:SM nanoemulsions and orthotopically implanted into the pancreas of NOD.SCID mice to assess their growth and metastasis formation (Figure 9a). To facilitate their visualization and identification *in vivo*, PANC1 cells were infected with a GFP-encoding
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lentivirus, and CAFs with an mCherry-encoding lentivirus. Fluorescence microscopy revealed the spheroid structure, with CAFs/mCherry forming a core surrounded by PANC1/GFP cells, and non-stained macrophages preferentially localizing on the exterior of the spheroid (Figure 9b).

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Established spheroids were either treated (or not) with 1 mg/mL of VitE:SM nanoemulsions, the media was changed 48 hours later, and orthotopic implantation of disaggregated spheres was performed the next day.

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Implanted NOD.SCID were monitored until day 90 post-implantation (Figure 9c). Upon euthanasia, we observed a reduction in the primary tumor burden in mice implanted with VitE:SM treated compared to untreated spheroids (Figure 9d and figure S11b), which we confirmed at the level of pancreatic/tumor weight (Figure 9e). Flow cytometry analysis of cells extracted from digested tumors also revealed a reduction in the percentage of PANC1/GFP cells in tumors generated from treated spheroids (Figure 9f). Histological analysis of these tumors showed a higher proportion of tumoral area in control tumors versus tumors generated from treated spheroids, where areas of healthy pancreas were clearly visible (Figure 9g). At the level of metastases, we did not detect macroscopic liver metastases in either control or treated mice (Figure S11c). However, at the cellular level, we found that mice implanted with treated spheroids had a significant reduction in PANC1/GFP cells in the liver compared to controls, determined by flow cytometry (Figure 9h).

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To confirm this result, we performed qPCR for the human *GAPDH* gene to determine the presence of cells of human origin in the liver. In line with our flow cytometric analyses, we detected lower amplification of *GAPDH* in mice implanted with treated spheroids compared to mice implanted with non-treated spheroids (Figure 9i).

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To validate that the effects observed were at the macrophage level, a parallel experiment excluding macrophages from the PANC1/GFP:CAFs/mCherry spheroids was performed. Spheroids devoid of macrophages were treated with VitE:SM nanoemulsions under the same conditions as the previous experiment, orthotopically implanted in NOD.SCID mice, and euthanized on day 90 post-implantation (Figure S12a). Upon analysis, we observed no significant reduction in tumor weight (Figure S12b-c) nor in the level of liver metastasis. Importantly, the analysis of the liver revealed no significant decrease in percentage of PANC1/GFP nor in the level of human *GAPDH* between the two experimental groups (Figure S12d-e). As

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such, and in line with the final conclusions determined in the murine system, these findings support the
main conclusion that the effectiveness of the nanoemulsions therapy is dependent on targeting/repolarizing
macrophages.

Altogether, these findings offer promising support for the possible translatability of a VitE:SM-based lipid
nanoemulsions therapy for human PDAC and highlights the pivotal role of macrophages as the mediators of
the therapeutic effect of this nanosystem. The demonstrated effectiveness of our lipid nanosystem therapy
in a human model of pancreatic cancer reinforces the concept that a novel treatment strategy, serving also
as a drug delivery system, holds the potential to reduce tumor growth and prevent liver metastasis for PDAC
patients.

## 650 CONCLUSIONS

In summary, we have validated a new type of nanoparticle therapy, a synergistic VitE:SM nanoparticle
composition, that offers the possibility of delivering chemotherapeutics like the TGF-βR1 inhibitor, LY, with
the objective of reprogramming TAMs of the tumor microenvironment to prevent liver metastasis in PDAC.
Both *in vitro* and *in vivo* models have demonstrated that VitE:SM nanoemulsion treatment modifies TAMs to
an M0/M1 state, resulting in an immunostimulant effect that promotes the reduction of PDAC primary
tumor growth and liver metastasis. The translatability of these results to the human model holds great
promise for reinforcing this platform's potential use in the treatment of PDAC and potentially other
metastatic cancers like colorectal cancer.

659

## 660 DECLARATIONS

### 661 Ethics approval

All in vivo procedures in mice were conducted in accordance with protocols approved by the local Animal
Experimental Ethics Committee of the Instituto de Salud Carlos III (PA 34-2012) or the Use Committee for
664 Animal Care from the Universidad Autónoma de Madrid (UAM) (Ref# CEI-25-587) and the Comunidad de Madrid (PROEX 335/14 or 294/19).

666 Consent for publication

667 All authors agree with the publication.

668 Data availability

669 FASTQ RNAseq data files generated have been deposited in Sequence Read Archive (SRA) of the National Center for Biotechnology Information (NCBI) with accession number ID: PRJNA1099468. All data underlying the results are available as part of the article. Additional information will be available on request.

672 Competing interests

673 The authors declare the following competing financial interest(s): M.d.I.F. is the co-founder and CEO of DIVERSA Technologies SL. The remaining authors declare no conflict of interest.

675 Funding

676 This study was supported by the MICIU Joint Transnational Call for Proposals/PCI 2019-1 (PCI2019-103725; B.S.,Jr.) and the Instituto de Salud Carlos III (AC18/00107 M.d.I.F.) grants, both part of the ERA-NET
677 EURONANOMED III project PANIPAC (grant number JTC2018/041), and FIS grants PI18/00757 and PI21/01110 (B.S.,Jr.), all co-financed through the European Regional Development Fund (FEDER) "Una manera de hacer Europa"; a Coordinated grant (GC16173694BARB; B.S.,Jr.) from the Fundación Asociación Española Contra el Cáncer (AECC); and a Max Eder Fellowship of the German Cancer Aid (111746) (P.C.H.).
678 A.P.-C. was supported by a Juan de la Cierva Formación Fellowship (FJC2020-044220-I). M.A.-S was supported by an IDIS Predoctoral Fellowship. J.C.L.-G. received support from a 'la Caixa' Foundation (ID 100010434) fellowship (LCF/BQ/DR21/11880011) and an EMBO Scientific Exchange Fellowship (ID 10210).
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684

685 Author contributions
A. P.-C., L. R.-C., M. A.-S. and J.C. L.-G.: Conceptualization, Investigation, Data extraction, Methodology, Formal analysis, Writing main manuscript text. S. B.-R., S. M. S., B. Y., D. N., S. A., J. A. R. and N. B.: Data extraction, Methodology, Formal analysis. L. S., S. M. and P. C. H.: Supervision. M.d.I.F. and B.S.,Jr.: Conceptualization, Writing-Review&Editing, Supervision, Project administration and obtained funding. All authors reviewed the manuscript.

691 Acknowledgements

We acknowledge Adrián Povo Retana for providing excess human PBMC samples and Antonio Castrillo for the IBMDM cell line.

694 FIGURE FOOTNOTES

**Figure 1.** Development and characterization of nanosystems. **a)** Schematic representation depicting the process of generating nanoemulsions through the ethanol injection method. Briefly, oils (VitE and/or OA and M) and surfactants (SM and/or PC, PA, PI, PS and PG) were mixed in an ethanolic organic phase (step 1). Subsequently, this mixture was injected in ultrapure water (step 2) and the diverse nanosystems were spontaneously formed (step 3) at room temperature. **b)** Summary of the components that make up the lipid nanosystems. Abbreviations, VitE: Vitamin E, SM: Sphingomyelin, OA: Oleic acid, M: Mygliol, PC: Phosphatidylcholine, PI: Phosphatidylinositol, PS: Phosphatidylserine, PG: Phosphatidylglycerol, PA: Palmitic acid.

**Figure 2.** The specific lipid composition of the nanosystems has an influence on M2 macrophage reprogramming. **a)** Experimental design for macrophage polarization and treatment with the lipid nanosystems: Immortalized murine Bone Marrow-Derived Macrophages (IBMDM) were induced to an M1 phenotype using LPS (10 ng/mL) + IFN-γ (10 ng/mL) or to an M2 phenotype with IL4 (10 ng/mL) for 24 hours. Following polarization, lipid nanosystems (1 mg/mL) were incubated with macrophages for a 4-hour duration: VitE:SM, M:SM, VitE:PC, VitE:SM:PI, VitE:SM:PS, VitE:SM:PG, and VitE:SM:PA at different concentrations of PA. Subsequently, IBMDM cells were washed and change to RPMI medium for qRT-PCR
and toxicity assays 24 hours later, or Western blot analyses 48 hours later. b) Relative luciferase activity (Fold change) (i.e., toxicity) ± SD determined in IBMDM cultures treated with the indicated different nanoemulsion compositions compared to untreated M2 polarized macrophages (n = 3). c) Analysis of the levels of the principal M2 marker Arg1 by qRT-PCR. Bars represent the mean fold change ± SD (n = 3), with untreated M2 set as 1.0. d) Top: Representative Western immunoblots of ARG1 protein expression levels. Bottom: Densitometric analysis of the immunoblots is represented in the bar diagram. Bars represent the mean fold change ± SD (n = 4), with untreated M2 set as 1.0. e) Summary table of three parameters chosen for evaluating of the best nanoemulsions composition. For toxicity, Low<4 AU, medium=4-7 AU, high>7 AU. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001; ns=not significant. One-way ANOVA test for multiple comparisons with Dunnett's post hoc test, compared to the untreated M2 sample.

**Figure 3. RNAseq analysis of M2 and M2 VitE:SM nanoemulsion-treated macrophages.** a) Volcano plot showing differentially expressed genes in VitE:SM nanoemulsions-treated M2 macrophages versus naïve M2 macrophages. Red dots, genes upregulated; blue dots, genes downregulated. Genes of interested have been labelled in black. b) Gene Set Enrichment Analysis (GSEA) plots showing a significant enrichment in the indicated gene signatures for VitE:SM nanoemulsions-treated M2 macrophages. Normalized Enrichment Score (NES) and p- and q- values are indicated for each plot. c) Histogram representation of the NES of significantly altered gene signatures down- (blue) and upregulated (red) in VitE:SM treated M2 macrophages.

**Figure 4. Intraperitoneal injection of VitE:SM nanoemulsions reduces tumor burden in an orthotopic KPC tumor model.** a) Experimental set-up for the *in vivo* syngeneic model in C57BL/6. Mice were orthotopically implanted with 2,500 KPC cells/pancreas and treatment was initiated 7 days post-implantation, receiving VitE:SM nanoemulsions treatments (69 mg/kg) 5 days per week for three weeks via intraperitoneal (IP) injection. b) Fold change ± SD in the total weight of the animals at the start and completion of the treatment. The initial weights for the control (n=10) and VitE:SM (n=9) groups from two independent experiments were pooled and each set as 1.0 to observe the weight evolution throughout the experiment. Days post-implant (DPI). c) Representative images of the liver at the experimental endpoint from control
736

(n=10) and VitE:SM-treated (n=9) groups from two independent experiments. Scale=1cm. d) Liver weight

737

from d). Data were normalized for each independent experiment (n=2) with the bars representing the mean

738

fold change ± SD, with the control set as 1.0. Unpaired t test. ns=not significant. e) Representative images

739

(40X) of H&E-stained liver slices at the experimental euthanize time point from control or VitE:SM-treated

740

mice. Zoom areas are depicted within squares. CV= Central vein, S= Sinusoids. Scales=400µm and 100µm. f) Representative images of the tumor (T) and adjacent peritoneum tumor-derived masses (PM) at the

741

experimental endpoint for control (n=10) and VitE:SM-treated (n=9) groups from two independent

742

experiments. Additional control with non-effective nanoparticles M:SM (n=4) was added. All tumor images

743

are presented in Figure S6. Scale=1cm. S=Spleen served as an anatomical reference for the pancreas. g) Fold

744

change in T + adjacent PM weight ± SD from g). Data were normalized for each duplicate experiment with

745

the control set as 1.0. One-way ANOVA with Dunnett's post hoc test, compared to control. ** = p < 0.01.

746

ns=not significant.

747

748

**Figure 5. Targeting capacity of the TopFluor® (TF) labelled VitE:SM nanoemulsions in vitro and in vivo. a)**

749

TopFluor® was incorporated in the surfactant (TopFluor®-labelled-sphingomyelin) and injected in the

750

aqueous phase as previously explained (Figure 1a). VitE:SM:TF= Vitamin E + TopFluor®-labelled-

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sphingomyelin nanosystem. b) Confocal images of BMDM cells treated or not with TF-labelled

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nanoemulsions (VitE:SM:TF, 0.5 mg/mL). Scale bar: 20 µM. DAPI: blue, TF: green. c) Cytometry dot plot of

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IBMDM cells in the presence or absence of VitE:SM:TF (0.5 mg/mL) nanoemulsions. IBMDMs containing

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VitE:SM:TF nanoemulsions were detected at a wavelength of Ex488nm Em530/30nm. SSC-A: Side Scatter

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(Area). d) Left: Flow cytometry analysis of the percentage of TopFluor®-positive live cells ± SD in three

756

different organs (liver, tumor, and lung) under two treatment modalities: intraperitoneal and retro-orbital

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(n=2 mice per treatment). Right: Flow cytometry analysis of the percentage of TopFluor®-positive live cells ±

758

SD in the liver under two intravenous treatment modalities (tail vein injection and retro-orbital) compared to

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control (n=5 control, n=5 mice per treatment). * = p < 0.05; *** = p < 0.001. One-way ANOVA with Dunnett's

760

post hoc test, compared to retro-orbital condition. e) Schematic representation of the markers used to

761

identify macrophages: CD45+, CD11b+ and F4/80+. f) Flow cytometry analysis of the percentage of
762 macrophages (CD45+, CD11b+ and F480+) ± SD targeted by the VitE:SM:TF nanoemulsions over time, with
763 data collected at 1h, 4h, 24h and 72h.

764 **Figure 6. Testing VitE:SM nanoemulsions loaded with the TGF-βR1 inhibitor (LY2157299).** a) For LY loading, 765 the TGF-βR1 inhibitor was dissolved in the organic phase and injected in the aqueous phase as previously 766 explained (Figure 1a). VitE:SM= Vitamin E + sphingomyelin nanosystem; VitE:SM:LY= Vitamin E + 767 sphingomyelin + encapsulated TGF-βR1 inhibitor. b) Left: Western immunoblot analysis of ARG1 protein 768 levels in murine BMDMs polarized as in Figure 2a to M0, M1 or M2 and treated with empty or LY 769 encapsulated VitE:SM nanoemulsions at two different concentrations: 1 and 0.5 mg/mL. Right: 770 Densitometric analysis of the immunoblots, with the bars representing the mean fold change ± SD (n = 3), 771 with untreated M2 set as 1.0. c) Analysis by qRT-PCR of two M2 markers, *Arg1* and *Egr2*, in the same 772 samples as described in b). Bars represent the mean fold change ± SD (n = 3), with untreated M2 set as 1.0. 773 d) Analysis by qRT-PCR of M1 markers, *Cd86*, *Il12b* and *Il1b*, in the same samples as in b) and c). Bars 774 represent the mean fold-change ± SD (n = 3), with untreated M1 set as 1.0. * = p < 0.05; ** = p < 0.01; *** = 775 p < 0.001; **** = p < 0.0001; ns=not significant. One-way ANOVA with Dunnett's post hoc test, compared to 776 M2 in b) and c) and to M1 in d).

777 **Figure 7. TGF-βR1 inhibitor (LY2157299)-loaded VitE:SM nanoemulsions reduce tumor growth and 778 diminishes TAM liver infiltration.** a) Schematic representation of the retro-orbital *in vivo* treatment 779 experiment. C57BL/6 mice were orthotopically implanted with 2,500 KPC cells/pancreas and retro-orbital 780 treatment was initiated on day 4 post-implantation and administered 3 days per week until the euthanize 781 points: 21, 28, or 35 days post-implant. b) Representative macroscopic images of tumors and adjacent PM 782 for the three groups (Control, VitE:SM:TF and VitE:SM:LY:TF) at the euthanize points: 21, 28, and 35 days 783 post-implant (DPI). Scale=1cm. c) (Left panel) Weight time course of tumors and adjacent PM from b) at the 784 three experimental end points. Three animals per group (n=3) were euthanized at 21 and 28 DPI and four 785 (n=4) at 35 DPI. (Right panel) Fold change in tumor and adjacent PM weight (g) for all animals across the 786 entire experimental time course. Bars represent the mean fold change ± SD (n = 10), with control set as 1.0. 787 One-way ANOVA test for multiple comparisons with Dunnett's post hoc test, compared to Control. d) 788
Representative images (40X) of H&E-stained tumors from control, VitE:SM:TF- or VitE:SM:LY:TF-treated mice on 35 DPI. T=Tumor, P=Pancreas. Scale=400µm. e) (Left panel) Ratio of the % area of tumor/healthy pancreas compared to the total histologic pancreas/tumor area quantified in the H&E slides from d). One-way ANOVA test for multiple comparisons with Dunnett's post hoc test, compared to Control. (Right panel) Frequency of samples from d) classified based on different percentages of tumor/healthy pancreas as defined in the legend. f) Flow cytometry analysis of the percentage of CD45+<CD11b+<F480+ macrophages in the liver samples from c. (Left panel) Time course of macrophage infiltration in the liver at the three euthanize points: 21, 28 or 35 DPI. One-way ANOVA test for multiple comparisons with Dunnett's post hoc test, compared to Control. (Right panel) Fold change in liver macrophage infiltration for all animals in the experiment. Bars represent the mean fold-change ± SD (n = 10), with Control set as 1.0. One-way ANOVA test for multiple comparisons with Dunnett's post hoc test, compared to Control. g) Flow cytometry analysis of the M2 TAM marker CD206. Bars represent the mean fold-change ± SD (n = 10), with Control set as 1.0. One-way ANOVA test for multiple comparisons with Dunnett's post hoc test, compared to Control. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001; ns=not significant.

**Figure 8.** TGF-βR1 inhibitor (LY2157299)-loaded VitE:SM nanoemulsions diminish liver metastasis in an intrasplenic KPC metastasis model. **a)** Schematic of the liver metastasis model involving the injection of KPC/mCherry cells into the spleen before organ resection using a cauterizer (C). **L=Liver. S=Spleen. SV: Splenic Vein.** **b)** Schematic representation of the intervention schedules in the liver metastasis model experiment. Spleens were injected with 25,000 KPC/mCherry cells, and retro-orbital VitE:SM nanoemulsions treatments were initiated on day 2 post-intrasplenic injection and continued for 16 days, administering three doses (25 mg/kg) per week. **c)** Weight of the liver (left) or Tumor + adjacent PM (right) ± SD. Data were collected from two independent experiments; Control (n=11) and VitE:SM:LY:TF (n=10). **d)** Representative macroscopic liver images from c). An arrow marks the metastasis in the liver. **Scale=1cm.** **e)** Representative H&E staining from samples obtained from the right lobe of the liver from d). Zoom areas are depicted within squares. **CV= Central vein, S= Sinusoids. Scales=400µm and 100µm.** **f)** Percentage of KPC/mCherry cells infiltrated in the liver. Bars represent the mean fold change ± SD, with Control set as 1.0. **g)** Densitometric
analysis of CRE/Gapdh PCR from Figure S8. Murine Gapdh was used as a housekeeping control. Bars represent the mean fold change ± SD, with Control set as 1.0. h) Flow cytometry analysis of the M2 TAM marker CD206 within the CD11b+F480+ population. Bars represent the mean fold change ± SD, with Control set as 1.0. Unpaired t test * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001; ns=not significant.

**Figure 9. Ex vivo treatment of human 3D tumor/CAF/macrophage spheroids with VitE:SM nanoemulsions.**

a) Schematic of human 3D tumor/CAF/macrophage spheroid generation, illustrating the process of generating human spheroids, comprising three populations of human cells: pancreatic cancer cells (PANC1), cancer-associated fibroblasts (CAFs) and macrophages. The 3D model was created as follows: on day one, PANC1/GFP and CAFs/mCherry were seeded in a non-adherent plate, and on day 3, macrophages were added. Spheroids were treated with VitE:SM nanoemulsions on day 4, the medium was changed on day 6, and on day 7, the spheroids were disaggregated and implanted orthotopically in the NOD.SCID mice. Mice were monitored for 90 days. b) Representative fluorescence microscopy image of a human spheroid co-culture. Scale=1mm. c) Schematic representation of the *in vivo* orthotopic human spheroid model in NOD.SCID mice. Mice were euthanized at 90 days post-implantation. d) Representative macroscopic photos of the tumors at the experimental end point (90 days post-implantation). S=Spleen served as an anatomical reference for the pancreas. Scale=1cm. e) Bar graph ± SD of tumor weight from d). Data compiled from two independent experiments (n=2) were normalized, and the control was set as 1.0. Unpaired t test. * = p < 0.05. f) Percentage of GFP+ cells (PANC1/GFP) ± SD in the tumor. Data were normalized for two independent experiments (n=2), and the control was set as 1.0. Unpaired t test. * = p < 0.05. g) Left: Representative images (40X) of H&E-stained tumor samples from d) are shown. T=Tumor, P=Pancreas. Scale=400µm. Right: Ratio of the % area of tumor/healthy pancreas ± SD compared to the total histologic pancreas/tumor area quantified in the H&E slides from the left panel. Unpaired t test. ns = not-significant. h) Percentage of GFP+ cells (PANC1/GFP) infiltrated in the liver. Bars represent the mean fold change ± SD, with Control set as 1.0. Unpaired t test. *** = p < 0.001. i) qRT-PCR analysis of the ratio of the human *hGAPDH* gene/murine beta actin (*mActb*). Bars represent the mean fold change ± SD, (n=2 independent experiments), with Control set as 1.0. Unpaired t test. * = p < 0.05.
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