Imaging and targeting S1PR1 in HER2+ tumors.

1.
Introduction
Gastric cancer remains a global health concern and ranks among the top five most frequently diagnosed cancers [1]. Gastric tumors are characterized by inter- and intra-tumor heterogeneity, which contributes to high relapse rates and frequent resistance to therapies used in the first-line treatment [2–5]. Gastric tumor resistance extends to tumors harboring amplifications or mutations in ERBB2, the gene encoding human epidermal growth factor receptor 2 (HER2) [6,7]. HER2-targeted therapies, including the monoclonal antibody trastuzumab and the antibody-drug conjugate (ADC) trastuzumab deruxtecan (T-DXd) [8,9], have shown clinical benefit in HER2-positive gastric cancer and have received regulatory approval (reviewed in [7]). However, beyond the observed efficacy of trastuzumab and T-DXd, clinical trials investigating other HER2-targeting antibodies have failed to demonstrate therapeutic benefits in patients with gastric cancer [10–12]. The limitations of gastric tumor response to HER2-targeted therapies highlight the need to uncover molecular pathways that influence therapeutic response and may serve as novel biomarkers and therapeutic targets in gastric cancer.
Sphingosine-1-phosphate receptor 1 (S1PR1) is a G-protein-coupled receptor that is expressed in different cell types, including tumor cells [13]. S1PR1 is activated by the signaling lipid sphingosine-1-phosphate (S1P), which leads to cell proliferation, migration, and survival [14–16]. Several strategies are currently under investigation to target the S1P signaling axis, including S1P-neutralizing antibodies, S1PR1 antagonists, and small-molecule inhibitors [14–16]. In gastric cancer, increased S1PR1 expression has been associated with drug resistance and poor prognosis in clinical samples [17,18]. Additionally, crosstalk between S1P receptors and receptor tyrosine kinases, including HER2 [19,20], plays a critical role in oncogenic signal transduction and therapeutic resistance. In this context, activation of S1PR1 has been shown to induce transactivation of HER2, which leads to HER2 tyrosine phosphorylation and amplification of downstream oncogenic signaling pathways [20]. These previous findings suggest that S1PR1 may serve as a modulator of HER2 signaling and a potential target to overcome resistance to HER2-directed therapies in gastric cancer.
S1PR1 modulators are FDA-approved for the treatment of multiple sclerosis [21], and we have developed S1PR1-targeted positron emission tomography (PET) tracers for noninvasive imaging of S1PR1 in multiple sclerosis, Alzheimer’s Disease, Parkinson’s Disease, and cerebral small vessel disease [22,23] (ClinicalTrials.gov IDs; NCT04517552, NCT06084533). With the USA FDA approval of the exploratory Investigational New Drug (e-IND), [11C]CS1P1 has been shown to be safe in humans [24]. In addition to carbon-11 tracers, we have developed fluorine-18 versions, which enable S1PR1-targeted radiotracers with slightly longer half-life, and enable multiple synthesis steps and extended imaging time points [25]. Building on our prior work, we apply S1PR1-targeted molecular imaging in the present work, leveraging S1PR1 PET in the context of tumor response to HER2-targeted therapy.
In this study, we investigated the relationship between S1PR1 expression and tumor response to HER2-targeted therapies. We combined immunohistochemistry, Western blot analyses, and S1PR1-targeted molecular imaging [24,26] to assess S1PR1 protein levels in patient-derived xenograft samples and preclinical models. Additionally, we evaluated the therapeutic impact of dual targeting HER2 and S1PR1 in vivo to explore potential synergism with this novel combinatorial strategy.

2.
Materials and methods
2.1.
Cell culture
NCIN87 (human gastric cancer cells, RRID:CVCL 1603) and SKHEP1 (endothelial cells that were isolated from the liver of a patient with adenocarcinoma, RRID:CVCL_0525) were purchased from the American Type Culture Collection. Human embryonic kidney 293 cells (HEK 293, RRID:CVCL_0045) were a gift from the Lewis Lab at MSKCC. Human peripheral blood mononuclear cells (PBMCs) were obtained from STEMCELL Technologies.
Culture media for NCIN87 cells includes Roswell Park Memorial Institute Medium (RPMI) 1640 growth medium supplemented with 10 % (v/v) fetal calf serum, 2 mM L-glutamine, 10 mM hydroxyethyl piperazineethanesulfonic acid, 1 mM sodium pyruvate, 4500 mg/L glucose, 1500 mg/L sodium bicarbonate, and 100 units/mL penicillin and streptomycin.
Culture media for SKHEP1 and HEK 293 cells consists of Minimum Essential Medium (MEM) with L-glutamine growth medium, supplemented with 10 % (v/v) fetal calf serum, 1500 mg/L sodium bicarbonate, 1 mM sodium pyruvate, non-essential amino acids, and 100 units/mL penicillin and streptomycin.
The cell lines used in this study were cultured in a humidified cell culture incubator at 37 °C and 5 % CO2.

2.2.
Generation of SKHEP1 Cells Stably Expressing HER2 (SKHEP1-hHER2)
SKHEP1-hHER2 cell line was generated using our previously reported protocols [27]. Briefly, SKHEP1 cells were transduced using 8 μg/mL of hexadimethrine bromide (Sigma) overnight. At 24 h after transduction, the cell culture media was changed. At 4 days after transduction, puromycin (1 μg/mL) was added for selection of SKHEP1-hHER2 cells. The selection using puromycin was continued for 6 days. Following transfection, the expression of HER2 was confirmed by Western blot.

2.3.
Western Blot
Total protein extracts were collected from the cells and tumor lysates. Cell lysates were prepared by placing cells on ice and performing cell scraping in radioimmunoprecipitation assay (RIPA) buffer (150 mM sodium chloride, 50 mM Tris hydrochloride [pH 7.5], 5 mM ethylene glycol tetraacetic acid, 1 % triton X-100, 0.5 % sodium deoxycholate, 0.1 % Sodium dodecyl sulfate, 2 mM phenylmethanesulfonyl fluoride, 2 mM iodoacetamide, and 1 protease inhibitor cocktail tablet). Tumor tissues were homogenized in RIPA buffer and all subsequent steps were performed in the same manner for both tumor and cell lysates.
Samples were then kept on ice and vortexed every 5 min for a total of 30 min, followed by centrifugation at 18,000 xg for 16 min at 4 °C. Supernatants containing total protein extracts were collected, followed by protein quantification using the Pierce Bicinchoninic Acid protein assay kit (Thermo Fisher Scientific). After protein quantification, the samples were mixed with NuPAGE lithium dodecyl sulfate sample buffer (Thermo Fisher Scientific) and denatured at 95 °C for 10 mins. Gel electrophoresis was conducted on the denatured samples using SDS-PAGE gels (NuPAGE 4–12 % Bis-Tris protein gels, Invitrogen), and then transferred to polyvinylidene difluoride membrane (Bio-Rad). Membranes were blocked for 1 h at room temperature in 5 % (w/v) milk (Bio-Rad) or bovine serum albumin (Sigma) prepared in tris-buffered saline buffer-saline containing 0.1 % (v/v) Tween 20 (TBS-T, EZ Bio-Research). Membranes were then incubated overnight with primary antibodies: mouse anti-β-actin 1:10,000 (A1978, Sigma, RRID: AB_476692), rabbit anti-HER2 1:800 (ab131490, Abcam, RRID:AB_11157090), and rabbit anti-S1PR1 1:200 (ASR-011, Alomone labs, RRID:AB_2039836). After incubation with the primary antibodies, the membranes were washed with TBS-T and then incubated with secondary antibody at room temperature for 1 h, goat anti-rabbit conjugated with AlexaFluor 680 (Invitrogen) or goat anti-mouse conjugated with AlexaFluor 800 (Invitrogen). Membranes were then washed again and scanned on the Odyssey infrared imaging system (LI-COR Biosciences, RRID:SCR_014579). Bands on images were analyzed using ImageJ/FIJI (RRID:SCR_002285).

2.4.
Radiosynthesis of [11C]-CS1P1 and [18F]-FS1P1
The radiotracers [11C]CS1P1 and [18F]FS1P1 share the same molecular structure, differing only in their radioisotopes: carbon-11 and fluorine-18, respectively. The radiosynthesis of [11C]CS1P1 and [18F] FS1P1 was achieved according to our previous procedure [25,28,29]. In brief, [11C]CS1P1 was prepared by the corresponding amino t-butyl ester precursor, which was treated with [11C]CH3OTf in acetonitrile and then deprotected the t-butyl group by hydrochloric acid, to afford [11C] CS1P1 with a radiochemical yield of ~60 %, high chemical purity and radiochemical purity (>98 %), and high molar activity of (>152 GBq/μmol, decay corrected to the end of bombardment [EOB]). [18F]FS1P1 was synthesized by nucleophilic radiofluorination of 2-nitro arylaldehyde precursor with TMEDA (N,N,N′,N′-tetramethylethylenediamine) as additive, followed by reductive amination and hydrolysis to yield [18F] FS1P1 with ~30 % radiochemical yield, >95 % radiochemical purity, and high molar activity (>74 GBq/μmol, decay corrected to end of synthesis [EOS]). Additional details are in Supplementary Methods.

2.5.
Binding studies
For the binding assays, solutions of [18F]-FS1P1 were prepared in PBS (pH 7.5) containing 1 % w/v human serum albumin (HSA, Sigma) and 0.1 % w/v sodium azide (NaN3, Acros Organics). Cells (1 million) were incubated with 10 μCi of [18F]-FS1P for 1 h at room temperature on a rotating platform. Unbound radioactivity was removed, and cells were washed twice with PBS by centrifugation. The pellet-bound radioactivity was measured on a gamma counter calibrated for fluorine-18.

2.6.
Autoradiography and S1PR1 immunohistochemistry
De-identified gastric patient-derived xenograft (PDX) tissues and their respective IHC scores were obtained from the Antitumor Assessment Core at Memorial Sloan Kettering Cancer Center through a material transfer agreement (MTA-Out00001384, RRID:SCR_012559). HER2-positivity is defined as IHC 3+, IHC 2+ and HER2:CEP17 FISH ratio ≥ 2.0, or ERBB2 amplification by next-generation sequencing. In vitro autoradiography study was carried out using [18F]FS1P1 in PDX tissues. In brief, tissues were pre-incubated with autoradiography buffer containing 10 mM HEPES, 5 mM MgCl2, 0.2 % BSA, and 0.1 mM EDTA in HBSS at pH 7.4 for 5 min. Tissues were then incubated with ~0.74 MBq of [18F]FS1P1 in buffer for 1 h at RT with gentle shaking, washed with buffer for 5 min three times, and then air dried for ~30 min. Dried slides were incubated with the BAS Storage Phosphor Screen (Care-stream, Rochester, NY) in a hypercassette autoradiography cassette (Cytiva, Amersham, UK) overnight. The autoradiography signal was quantified using a Typhoon FLA 9000 phosphor imaging system (GE Healthcare, Chicago, IL). To determine non-specific binding, 10 μM of S1PR1-specific antagonist, NIBR-0213 (Cayman, Ann Arbor, MI), was used. All images were processed and analyzed using Fiji ImageJ (RRID: SCR_002285), and all representative images were processed using a continuous lut “Physics.”
Immunohistochemistry (IHC) was performed using standard IHC methods. All sections were pre-warmed at room temperature and fixed with 4 % paraformaldehyde in PBS for 10 min and then washed with PBS three times. Sections were then incubated with ReadyProbes Endogenous HRP and AP Blocking Solution (ThermoFisher, Waltham, MA) for 10 min, followed by blocking with 5 % horse serum for 2 h. All sections were then incubated with anti-S1PR1 antibody (Proteintech, Rosemont, IL, RRID:AB_10793721) at 4 °C overnight and then washed in PBS three times. Sections were then incubated with HRP-conjugated goat anti-rabbit IgG antibody for 1 h and developed using ImmPACT DAB (Vector Laboratories, Burlingame, CA, RRID:AB_2336520). Slides were scanned automatically using a Zeiss Axio Scan.Z1 (Carl Zeiss, Jena, Germany, RRID:SCR_020927) and analyzed using ImageJ (RRID: SCR_002285).

2.7.
Animal studies and tumor xenografts
All animal experiments were conducted following the guidelines provided by the Research Animal Resource Center and Institutional Animal Care and Use Committee at Washington University School of Medicine at St. Louis. Mice were housed in type II polycarbonate cages, fed with a sterilized standard laboratory diet, and given sterile water ad libitum. Housing conditions were maintained around 19 °C–23 °C, at 30 %–70 % relative humidity, and in a 12 h light/12 h dark cycle.
Tumor measurements were determined using an external vernier caliper twice per week. Subsequently, tumor volume (V, mm3) was calculated using the eq. V = (4π/3) × (α/2)2 × (b/2),where α (mm) is the longest axis, and b (mm) is the axis perpendicular to the longest axis.
2.7.1.
NCIN87 xenografts
Eight- to 10-week-old nu/nu female mice (Charles River Laboratories, RRID:SCR_003792) were xenografted with 5 × 106 NCIN87 cells in a 100-μL cell suspension of a 1:1 (v/v) mixture of matrigel (BD Biosciences). Tumors were monitored until they reached approximately 100 mm3 as measured by an external vernier caliper.

2.7.2.
SKHEP1-hHER2 xenografts
Eight- to 10-week-old nu/nu female mice (Charles River Laboratories, RRID:SCR_003792) were xenografted with 1 × 106 SKHEP1-hHER2 cells in a 100-μL cell suspension of a 1:1 (v/v) mixture of matrigel (BD Biosciences). Tumors were monitored until they reached approximately 100 mm3 - 200 mm3 as measured by an external vernier caliper.

2.8.
Positron emission tomography (PET) imaging and biodistribution
PET/CT imaging was performed on a Mediso nanoScan PET/CT scanner (Mediso). Mice were anesthetized 10 min before the PET scanner start time, by inhalation of 1.5 %–2 % isoflurane (Baxter Healthcare) in an oxygen gas mixture. PET imaging data for each group were recorded with the mice under isoflurane anesthesia (1.5 %–2 %). CT scans were acquired for 5 min, followed by a 20-min PET scan. The images were analyzed using Imalytics Preclinical software (version 3.1, Gremse-IT23, http://exmi.rwth-aachen.de/, RRID:SCR_026980) or ASIPro VM software (Concorde Microsystems, RRID:SCR_014547). The tumor was extracted by using the shape from the CT to outline the tumor, and then, once the CT was extracted, the CT image was overlaid on the PET signal in the tumor.
Biodistribution studies were performed post-PET imaging acquisition. The mice were sacrificed by controlled carbon dioxide overdose followed by cervical dislocation, and organs were harvested and measured in the γ-counter. Radioactivity associated with each organ was expressed as percentage injected dose per gram of organ (%ID/g).
After biodistribution, autoradiography was performed on tumor tissues placed in a film cassette against phosphor imaging plate (Fujifilm BAS-MS2325; Fuji Photo Film) at −20 °C. Phosphor imaging plates were read at a pixel resolution of 25 μm with a Typhoon 7000 IP plate reader (GE Healthcare). Quantification on autoradiography was performed using ImageJ, by selecting regions of interest.

2.9.
Therapeutic studies
2.9.1.
Trastuzumab therapy
When HER2+ NCIN87 gastric tumors reached 100–300 mm3, the mice were treated with trastuzumab or vehicle (n = 5 mice in each group), following our previously reported methods [27]. Trastuzumab was administered at 5 mg/kg (intraperitoneal, weekly for 5 weeks).

2.9.2.
T-DXd therapy
Once SKHEP1-hHER2 xenografts reached approximately 200 mm3, mice were divided into groups of n = 10 mice in each group: saline, fingolimod, T-DXd, or a combination of fingolimod (AB142065, abcam) and T-DXd. Fingolimod was administered at 5 mg/kg (intraperitoneal) and T-DXd was administered by intravenous injection at 5 mg/kg. Mice receive two doses of fingolimod: at the same time as the intravenous injection of T-DXd, and 12 h after T-DXd. At 20 days after initial treatment, mice were treated a second time with T-DXd and/or fingolimod.

3.
Results
3.1.
S1PR1 protein levels in HER2-positive gastric tumor samples
Previous studies have shown that S1PR1 transcript and protein levels are elevated in advanced-stage gastric cancer and in tumors resistant to chemotherapy compared to those from patients with complete remission [18]. In our study, we first explored S1PR1 protein levels in HER2+ gastric tumor patient-derived xenografts (PDXs) using Western blot and IHC. S1PR1 protein levels were variable across different tumor samples (Fig. 1A). Next, we used an S1PR1-targeting imaging agent (Supplementary Fig. 1), [18F]-FS1P1 [26], to detect S1PR1 in HER2+ PDXs by autoradiography. Autoradiography demonstrated S1PR1 presence in the tumor samples and the uptake was blocked with an excess of S1P1 (10 μM NIBR-0213), indicating specific binging of [18F]-FS1P1 to S1PR1 on the tumor samples (Fig. 1A).
These data indicate the presence of S1PR1 in HER2+ gastric tumor samples.

3.2.
S1PR1 protein levels in tumors treated with trastuzumab
Previous work has shown that S1P induces phosphorylation of HER2 in gastric cancer, leading to further downstream signaling [20]. We compared S1PR1 protein levels in NCIN87 HER2+ gastric tumors that responded to HER2-targeting trastuzumab therapy versus tumors that showed resistance to trastuzumab. Mice bearing HER2+ NCIN87 tumors were treated with an intraperitoneal injection of trastuzumab at 5 mg/kg weekly over 5 weeks [30]. At 60 days after treatment initiation, response was defined by a tumor volume fold change (from therapy initiation) lower than 1. Mice whose tumors had a tumor volume fold change of ~1 (no increase or decrease from therapy initiation) were considered partial responders. Compared to the untreated control group, trastuzumab responders demonstrated a one-third reduction (**p < 0.01) in S1PR1 protein levels as assessed by Western blot. S1PR1 protein levels in partial responders were slightly lower than levels in control tumors, although the trend was not significant (Fig. 1B,C; Supplementary Figs. 2,3). IHC and [18F]-FS1P1 autoradiography confirmed lower S1PR1 protein levels in tumors that responded to trastuzumab therapy than in untreated control tumors (Fig. 1D).
Altogether, the Western blot, IHC, and autoradiography studies indicate that S1PR1 expression is decreased in HER2+ tumors that respond to trastuzumab.

3.3.
[18F]-FS1P1 uptake in HER2+/S1PR1+ tumors treated with T-DXd
The decrease in S1PR1 protein levels among trastuzumab-responding tumors suggest the potential of S1PR1 molecular imaging using [18F]FS1P1 or [11C]CS1P1 (Supplementary Fig. 1) to correlate with treatment outcomes. To test this, we transduced SKHEP1 cells with a lentivirus containing human HER2 (Fig. 2A, Supplementary Fig. 4), to generate a stably expressing HER2 cell line (SKHEP1-hHER2) that expresses S1PR1 at higher levels than NCIN87 gastric cancer cells. The parental line of SKHEP1 cells has previously been used to test S1PR1 radiotracers [31] and express S1PR1 at five-fold higher protein levels than NCIN87 cancer cells (Fig. 2A).
Biodistribution of radiolabeled S1P1 was determined in three HER2+ tumor models: NCIN87, SKHEP1-hHER2, and a HER2+ PDX (Supplementary Figs. 5, 6). A 1.15 ± 0.30 tumor-to-muscle ratio (Fig. 2B) of the radiotracer was observed in NCIN87 tumors. Notably, SKHEP1-hHER2 tumors demonstrated a higher tumor-to-muscle ratio of [18F]FS1P1 when compared with NCIN87 tumors or a patient-derived xenograft (*p < 0.05, Fig. 2B). Subsequent studies were, therefore, performed using the SKHEP1-hHER2 model.
To confirm that there were no effects from HER2 transduction on [18F]FS1P1 binding in the SKHEP1-hHER2 model, a radioligand binding assay was performed. There was no significant change in [18F]FS1P1 binding in the parental SKHEP1 cells compared with SKHEP1-hHER2 cells (Fig. 2C). Additionally, [18F]FS1P1 binding was similar between both SKHEP1 cell lines and peripheral blood mononuclear cells (PBMCs) obtained from a healthy donor, which include S1PR1(+) T cells [32], and was significantly higher than in non-cancer HEK 293 cells (***p < 0.001).
Following our results obtained in tumors from trastuzumab-treated mice in Fig. 1B showing a decrease in S1PR1 protein levels in HER2+ NCIN87 tumors that responded to anti-HER2 antibody trastuzumab, we next determined changes in S1PR1 protein levels in mice treated with the trastuzumab-drug conjugate T-DXd. A single dose of T-DXd (5 mg/kg, intravenous) was used in the therapeutic studies. Animals bearing SKHEP1-hHER2 tumors were treated with T-DXd and S1PR1 was monitored by ex vivo biodistribution, autoradiography, and Western blot analyses (Fig. 2D). Biodistribution and autoradiography of tumors treated with T-DXd indicated a 2-fold reduction (*p < 0.05) in S1PR1 protein levels when compared with the control saline cohort (Fig. 2E,F; Supplementary Fig. 7).
Tumors that responded to T-DXd (Fig. 3A) showed lower levels of S1PR1, as detected by Western blot of tumor lysates (Fig. 3B; Supplementary Fig. 8) and by [18F]FS1P1 PET imaging (Fig. 3C). Tumor to muscle ratio was ~5-fold higher (***p < 0.001) in non-responders when compared with T-DXd responders (Fig. 3D).

3.4.
Dual targeting of HER2 and S1PR1 with T-DXd plus Fingolimod
The negative relationship between S1PR1 protein levels and tumor response to trastuzumab or T-DXd suggests that dual targeting of both S1PR1 and HER2 proteins has potential as a novel combinatorial approach to improve tumor outcomes. To determine this, we utilized fingolimod, an analog of S1P, clinically used in the treatment of multiple sclerosis [21], in combination with T-DXd. Mice bearing HER2+/S1PR1+ SKHEP1-hHER2 tumors were administered intraperitoneal fingolimod at the same time as T-DXd injection and at 12 h after T-DXd (Fig. 4A). At 20 days after initial treatment, mice were treated a second time with T-DXd and/or fingolimod. Mice treated with fingolimod only showed a slight increase in tumor volume when compared with saline. Mice treated with either T-DXd alone or T-DXd in combination with fingolimod showed tumor growth inhibition. The therapeutic efficacy of T-DXd combined with fingolimod was significantly improved compared with T-DXd alone (Fig. 4B–D). Western blot analyses of excised tumors demonstrated higher depletion of HER2 and S1PR1 in the T-DXd/fingolimod group when compared with T-DXd alone (Fig. 4E; Supplementary Fig. 9). Together, these results show that the in vivo efficacy of T-DXd is improved when administered in combination with an S1PR1 inhibitor.

4.
Discussion
T-DXd has demonstrated clinical efficacy in HER2-positive gastric cancer, but a significant number of patients do not respond to treatment, and resistance remains a major challenge [7–9]. In this study, we determined the expression of S1PR1, a G-protein-coupled receptor known to modulate oncogenic signaling [13,18], in patient-derived gastric xenografts and in tumors treated with HER2-directed therapy. Our findings indicate that S1PR1 expression is associated with poor tumor response to HER2-targeted treatment, including trastuzumab and T-DXd, and that dual targeting of HER2 and S1PR1 enhances preclinical therapeutic efficacy.
Western blot, immunohistochemistry, and S1PR1-targeted molecular imaging demonstrated that S1PR1 protein levels were decreased in tumors that responded to HER2-targeted therapy, while tumors resistant to therapy exhibited high S1PR1 expression. These findings are consistent with previous work showing crosstalk between S1PR1 and HER2 signaling, where S1PR1 activation induces HER2 transactivation and amplifies downstream signaling pathways such as ERK and Akt [19,20]. This study further highlights the importance of the tumor microenvironment and alternative signaling axes (like S1PR1) in modulating the response to ADCs.
Our previous research efforts have focused on the development of S1PR1-targeting radioligands with improved pharmacokinetic and pharmacodynamic properties to enable clinical translation. Among these, two structurally identical radioligands, [11C]CS1P1 and [18F] FS1P1, have been synthesized using either carbon-11 or fluorine-18 isotopes, respectively [24,26]. Despite being labeled with different radionuclides, both tracers share the same molecular structure and exhibit comparable binding profiles to S1PR1 [26]. These radioligands have practical advantages for PET imaging: the short half-lives of carbon-11 (20.4 min) and fluorine-18 (109.7 min) result in low radiation exposure to subjects and enable the possibility of performing multiple imaging sessions within a single day. In particular, [18F]FS1P1 enables multicenter clinical trials, as fluorine-18 is the most widely used PET isotope and benefits from established infrastructure for radiolabeling and distribution across imaging centers. The ability of [18F]-FS1P1 to quantify S1PR1 in tumors has the potential to be a promising approach for stratifying tumors likely to respond to HER2-targeted treatment and monitoring S1PR1 dynamics in vivo. To further enhance imaging sensitivity and temporal resolution, ongoing studies in our group are focused on the development of the next-generation S1PR1-targeted compounds with improved tumor uptake and pharmacokinetics. In parallel, we are exploring radiolabeling with longer-lived isotopes, such as copper-64 (half-life: 12.7 h), to enable imaging at later time points and characterize tracer retention and distribution within tumors and peripheral tissues. Optimization of tumor uptake and distribution will further enable the use of S1PR1-targeted compounds as radiosensitizers or chemosensitizers in subsequent studies.
Overall, our study demonstrates the potential of S1PR1 as both a biomarker and a therapeutic target in HER2-positive tumors. Given that fingolimod has a known safety profile [21] and is approved for other indications, a prospective clinical study is needed to determine the safety and efficacy of T-DXd/fingolimod. Previous studies have demonstrated the therapeutic potential of S1PR1 modulation across various cancer types [15]. Hait et al. demonstrated that phosphorylated prodrug fingolimod acts as a histone deacetylase inhibitor and sensitizes triple-negative breast cancer to endocrine therapy, which further shows its dual mechanism of action in tumor suppression [33]. Additionally, Molho-Pessach et al. showed that S1PR1 signaling promotes tumor-infiltrating regulatory T-cell expansion and immune evasion in bladder cancer [34]. These findings, along with data from our current study, suggest the potential of S1PR1-targeted strategies (both as a monotherapy and in rational combination regimens) to improve therapeutic outcomes in cancer. Although fingolimod is clinically used in the treatment of multiple sclerosis [21], there are no approved uses of fingolimod or other S1P1 receptor modulators in patients with gastric cancer. Additional studies testing the combination of T-DXd with fingolimod are needed to inform their potential clinical combination.
Given that S1PR1-targeted PET imaging has already been evaluated in humans, future studies are well-positioned to translate this agent to the clinic, with the potential to extend its application beyond gastric cancer to other tumor types currently treated with T-DXd. In this context, T-DXd is FDA approved for breast, non-small cell lung cancer, colorectal cancer; and it is under investigation or off-label use for bladder, pancreatic, biliary tract, endometrial, salivary gland, ovarian, and prostate cancer. Future studies are needed to further investigate the mechanisms by which S1PR1 affects HER2 signaling and resistance. Temporal studies evaluating changes in S1PR1 and HER2 levels at different time points post-therapy will further determine optimal treatment schedules. In addition, PET imaging can be leveraged to titrate fingolimod dosing in preclinical models and identify the most effective dose combination with T-DXd. Investigating the effects of fingolimod and HER2-targeted therapy in HER2-low or ultra-low models, where ADC efficacy remains suboptimal [35], could also expand the utility of this approach.
Although our study provides preclinical evidence supporting the dual targeting of HER2 and S1PR1, it is limited by the use of immune-deficient mice and engineered tumor models. Additional validation is needed to confirm the translational potential of this strategy. Larger cohorts of S1PR1-positive tumor samples are also required to better define expression patterns and support the rationale for advancing these approaches into human studies. The use of T-DXd, which does not bind murine HER2, also limits the ability to assess off-target accumulation and toxicity in non-tumor tissues. As such, future work should assess systemic toxicity and biodistribution in models that recapitulate human HER2 expression patterns.
Overall, this study identifies a previously underappreciated link between S1PR1 expression and resistance to HER2-targeted therapy in gastric cancer. We demonstrate that S1PR1-targeted molecular imaging can be used to assess response and that pharmacologic inhibition of S1PR1 enhances the antitumor activity of T-DXd in HER2+/S1PR1+ tumors. These findings warrant further investigation, with the goal of improving therapeutic outcomes for patients with HER2-expressing gastric tumors through rational combination therapy.

5.
Conclusion
This study demonstrates that high S1PR1 expression is associated with reduced response to HER2-targeted therapies in gastric cancer. Using [18F]FS1P1, we detected differences in S1PR1 levels in tumors that responded or failed to respond to T-DXd. Dual targeting of HER2 and S1PR1 using T-DXd in combination with the S1PR1 modulator fingolimod enhanced treatment efficacy in HER2+/S1PR1+ xenografts and resulted in decreased tumor growth and downregulation of HER2 and S1PR1 expression. These findings highlight the potential of S1PR1 as both a therapeutic target and imaging biomarker in HER2-positive tumors. Future studies will optimize and evaluate the utility of S1PR1-targeted radiotracers for patient stratification and validate the safety and efficacy of HER2/S1PR1 combination therapy.

Supplementary Material
Supplementary Information