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. 2026 Aug 4:10.1111/cas.70489. Online ahead of print. doi: 10.1111/cas.70489

Targeted Photo‐Induced Elimination of Tumor‐Associated Macrophages Suppresses Cancer Progression in a Murine Model

Kohei Nakajima 1, Tomomi Uchiyama 1, Kazuki Funayama 1, Yuto Goto 1, Akari Mukaimine 2, Naoya Ieda 1, Mikako Ogawa 1,2,✉
PMCID: PMC13435534  PMID: 42549760

ABSTRACT

Tumor‐associated macrophages (TAMs) are key components of the immunosuppressive tumor microenvironment (TME) and represent attractive therapeutic targets to enhance antitumor immunity. However, systemic TAM‐depletion strategies targeting colony‐stimulating factor 1 receptor (CSF1R) are associated with the risk of off‐target toxicity due to the depletion of macrophages in healthy tissues. Here, we investigated the potential of near‐infrared photoimmunotherapy (NIR‐PIT) as a strategy for selective, photo‐induced elimination of TAMs. The Fab fragment of an anti‐CSF1R antibody was conjugated with the photosensitizer IRDye 700DX (IR700), and the therapeutic effects of the conjugate were evaluated in a murine cancer model. Upon NIR light irradiation, Fab‐IR700 significantly suppressed tumor growth and improved overall survival of the tumor‐bearing mice. Notably, repeated NIR‐PIT cycles enhanced therapeutic outcomes and induced durable anticancer effects, as indicated by the rejection of reimplanted cancer cells. Flow cytometry and histological analyses confirmed TAM depletion and significant alterations in the intra‐tumoral immune cell populations posttreatment. Overall, CSF1R‐targeted NIR‐PIT can effectively eliminate TAMs without systemic toxicity, thus presenting a promising strategy to remodel the TME toward an antitumor state and synergize with other immunotherapies against refractory tumors.

Keywords: colony‐stimulating factor 1 receptor (CSF1R), IRDye 700DX (IR700), near‐infrared photoimmunotherapy (NIR‐PIT), phototherapy, tumor‐associated macrophage (TAM)

This study demonstrates the selective depletion of tumor‐associated macrophages (TAMs) by targeting CSF1R through near‐infrared photoimmunotherapy (NIR‐PIT). Using a combination of anti‐CSF1R Fab‐IR700 conjugates and localized light irradiation, we achieved tumor suppression and durable antitumor immunity without systemic toxicity. This approach represents a new strategy to reprogram the tumor microenvironment and may synergize with existing immunotherapies for the treatment of refractory cancers.

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Abbreviations

ADCC

antibody‐dependent cellular cytotoxicity

CAFs

cancer‐associated fibroblasts

CSF1R

colony‐stimulating factor 1 receptor

CTLA4

cytotoxic T‐lymphocyte antigen‐4

EGFR

epidermal growth factor receptor

IR700

IRDye 700DX

MDSCs

myeloid‐derived suppressor cells

NK cells

natural killer cells

NIR‐PIT

near‐infrared photoimmunotherapy

PD‐1

programmed cell death‐1

TAM

tumor‐associated macrophage

TME

tumor microenvironment

Tregs

regulatory T cells

1. Introduction

The tumor microenvironment (TME) is a highly complex ecosystem consisting of stromal cells, immune cells, and cancer cells that interact with each other. The tumor‐associated macrophages (TAMs) are one of the key components of the TME, and may constitute up to or even more than 50% of the cells in solid tumors [1]. The TAMs are derived from circulating monocytes, which are recruited to the tumors and undergo reprogramming by cancer cells to support angiogenesis and metastasis, etc. [2]. In addition, TAMs play a crucial role in suppressing the function of CD8+ cytotoxic T lymphocytes and natural killer (NK) cells, which facilitates immune evasion of cancer cells [3, 4]. Thus, TAMs are an attractive target for therapies that modulate the antitumor immune response in solid tumors.

Studies show that depletion of TAMs can enhance antitumor immunity and improve the efficacy of immunotherapies. Colony‐stimulating factor 1 receptor (CSF1R) has recently attracted attention as a promising target for the depletion of TAMs [5, 6]. It is expressed on the surface of myeloid cells such as monocytes, macrophages, and microglia [7]. Furthermore, the pro‐tumorigenic M2 macrophages in the TME express high levels of CSF1R. Thus, targeting the intra‐tumoral CSF1R‐expressing cells may achieve tumor regression by reversing the immunosuppressive TME. In recent clinical trials, CSF1R‐specific monoclonal antibodies have exhibited therapeutic effects by depleting the TAMs and enhancing antitumor immune responses [5]. However, since monocytes and macrophages in healthy tissues also express CSF1R, systemic administration of anti‐CSF1R antibodies could also deplete these cells via antibody‐dependent cellular cytotoxicity (ADCC) and other mechanisms, resulting in immunosuppression or systemic toxicity [8, 9, 10]. Therefore, it is crucial to develop strategies that selectively eliminate CSF1R‐expressing cells within the TME without affecting the resident macrophages in normal tissues.

To address this issue, we focused on near‐infrared photoimmunotherapy (NIR‐PIT), a novel phototherapy that can selectively kill targeted cells within the irradiated tumor [11, 12]. NIR‐PIT utilizes conjugates of monoclonal antibodies and a photosensitizer such as IRDye 700DX (IR700). When stimulated with NIR light of wavelength 690 nm, the antibody‐IR700 conjugate can induce physical stress in the plasma membrane of target cells, eventually leading to cell death [13, 14]. Since NIR‐PIT does not depend on ADCC or other biological pathways such as signaling inhibition, antibody fragments that lack Fc regions can also be used for NIR‐PIT [11, 15]. In addition, NIR‐PIT enables precise and rapid elimination of targeted cells only within the light‐irradiated lesions, thereby minimizing damage to healthy tissues [12]. In this study, we explored the potential of NIR‐PIT for the photo‐induced elimination of TAMs by using a conjugate of IR700 and the Fab fragment of an anti‐CSF1R antibody.

2. Materials and Methods

2.1. Reagents

IRDye 700DX NHS ester, a water‐soluble, silicon‐phthalocyanine derivative, was obtained from Rakuten Medical Inc. (Tokyo, Japan). All other chemicals were of commercially available reagent grade.

2.2. Preparation of the Fab Fragment of Anti‐CSF1R Monoclonal Antibody

Anti‐CSF1R monoclonal antibody (Clone: AFS98) was purchased from BioXCell (West Lebanon, NH, USA). The Fab fragment was enzymatically cleaved from the antibody using papain immobilized on agarose resin (Thermo Fisher Scientific Inc., Rockford, IL, USA). Briefly, anti‐CSF1R antibody (1 mg, 6.67 nmol) and resin‐bound papain (250 μL of a commercial suspension) were mixed in 20 mM Na3PO4, 10 mM EDTA, and 20 mM cysteine (pH 7), and incubated at 37°C for 5 h with constant shaking. The supernatant was then loaded onto a HiTrap Protein G column (Cytiva, Global Life Sciences Technologies Japan K.K., Tokyo, Japan) to remove unbound Fab fragments. The Fc fragment was subsequently eluted using 0.1 M glycine‐HCl buffer (pH 2.7), and concentrated via ultrafiltration in PBS using an Amicon Ultra 10 K device (Merck Millipore, Billerica, MA, USA). The Fab fragment was analyzed by size‐exclusion HPLC using a COSMOSIL(R) 5Diol‐300‐II Packed Column (5 μm, 7.5 × 300 mm) (Nacalai Tesque, Kyoto, Japan) with 0.1 M phosphate buffer (pH 7).

2.3. Conjugation of IR700 With Anti‐CSF1R Fab Fragment

Fab fragments (1 mg, 20 nmol) and IR700 NHS ester (120 μg, 61.2 nmol) were mixed in 0.1 M Na2HPO4 buffer (pH 8.5) and incubated at room temperature for 3 h. The reaction mixture was purified using an Amicon Ultra 10 K device to obtain the Fab‐IR700 conjugates. The protein concentration was quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific Inc.) by measuring the absorbance at 562 nm with a microplate reader (Infinite M200; Tecan Austria GmbH). The concentration of IR700 was determined spectrophotometrically by measuring absorbance at 689 nm using a UV‐1850 spectrophotometer (Shimadzu Corp.). The conjugation ratio of IR700 to Fab fragments was calculated to be ~2.

2.4. Cell Culture

The murine colorectal cancer cell line MC38 was obtained from Kerafast Inc. (Boston, MA, USA). Mouse macrophage RAW264 was purchased from RIKEN BRC Cell Bank (Tsukuba, Japan). The MC38 cells were maintained in Dulbecco's Modified Eagle Medium (DMEM; Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (FBS; Gibco Life Technologies, Grand Island, NY, USA) and 1% penicillin–streptomycin (Nacalai Tesque) in a humidified incubator at 37°C with 5% CO2. The RAW264 cells were maintained in MEM (Sigma‐Aldrich, St. Louis, MO, USA) supplemented with 10% FBS, 1% penicillin–streptomycin, and 1% nonessential amino acids (NEAA; Nacalai Tesque). The cultures were regularly tested for mycoplasma infection using the TaKaRa PCR Mycoplasma Detection Set (Takara Bio Inc., Shiga, Japan).

2.5. In Vitro Microscopic Observations

MC38 cells or RAW264 cells were cultured alone after they were seeded at 2.0 × 105 cells per 35‐mm plastic dish. To distinguish MC38 cells, they were labeled with CellTrace CFSE Cell Proliferation Kit (Thermo Fisher Scientific Inc.) before the seeding. After 24 h incubation at 37°C, Fab‐IR700 was added to the culture medium at a final concentration of 1.7 μg/mL. Following an additional 6 h incubation, the medium was replaced with PBS, and fluorescence imaging was performed using a microscope (BX41, Evident, Tokyo, Japan). For coculture experiments, non‐labeled RAW264 cells (1.2 × 105 cells) and MC38 cells (1.5 × 105 cells) labeled with CellTrace CFSE Cell Proliferation Kit were seeded together in 35‐mm plastic dishes. After 24 h incubation at 37°C, Fab‐IR700 was added at a final concentration of 1.7 μg/mL and incubated for 6 h. The medium was then replaced with PBS supplemented with 2 μM ethidium homodimer‐1 (EthD‐1; Thermo Fisher Scientific Inc.). Pre‐images were obtained before NIR light irradiation. Cells were subsequently exposed to NIR light (673–748 nm) for 30 s, and postirradiation images were obtained 60 min later. Fluorescence was detected using appropriate filter sets: CellTrace (Ex: 460–490 nm, Em: > 520 nm), IR700 (Ex: 673–748 nm, Em: > 765–855 nm), and EthD‐1 (Ex: 480–550 nm, Em: > 590 nm).

2.6. Establishment of MC38 Tumor‐Bearing Mice

All in vivo experiments were performed in compliance with the Guidelines for the Care and Use of Laboratory Animals and approved by the Hokkaido University Animal Care Committee. Seven‐weeks‐old female C57BL/6JJmsSlc mice were obtained from Japan SLC Inc. (Shizuoka, Japan). The mice were anesthetized using 1.0%–1.5% isoflurane during all procedures. The mice were subcutaneously injected with 6 × 106 MC38 cells in 100 μL PBS into the left leg or bilaterally into both legs. In vivo NIR‐PIT experiments were conducted for 7–8 days postinoculation.

In the tumor rechallenge experiment, mice were first inoculated with MC38 cells to establish primary tumors. Following treatment with 4‐cycle NIR‐PIT group with 50 J/cm2, mice that achieved complete response were identified. On day 43 after the initial tumor inoculation, these CR mice were rechallenged by subcutaneous injection of MC38 cells into the contralateral leg (6 × 106 cells per mouse). For the control group, mice were maintained in the same animal facility under identical housing conditions but did not receive tumor inoculation at the initial time point. These mice were kept untreated until day 43. On day 43, control mice were subcutaneously inoculated with MC38 cells at the same dose (6 × 106 cells per mouse). Thus, in the present study, factors such as age, breeding period, and housing conditions were matched between the control and rechallenge NIR‐PIT groups.

2.7. In Vivo Fluorescence Imaging

Fab‐IR700 (6.67 μM, 100 μL) was intravenously administered into the tumor‐bearing mice, and fluorescence images were taken 0.5, 1, 3, 6, 24, 48, and 72 h postinjection using an IVIS Lumina LT Series III imaging system (PerkinElmer) equipped with a 650–670 nm excitation filter and a 690–730 nm emission filter. Regions of interest were defined on the tumor‐bearing left leg (tumor) and the non‐tumor‐bearing right leg (background). Both tumor and background signals were normalized by subtracting the noise signal, and the tumor‐to‐background ratio was calculated.

2.8. NIR‐ PIT Targeting CSF1R

Mice bearing MC38 tumors were intravenously injected with Fab‐IR700 (6.67 μM, 100 μL). At 6 h postinjection, tumors were irradiated with NIR light (MLL‐III‐690; Changchun New Industries Optoelectronics Tech Co. Ltd.) at a total dose of 50 J/cm2. The power density was determined to be 150 mW/cm2 using an optical power meter (PM121D; Thorlabs, Newton, NJ, USA). The duration of light irradiation was precisely 5 min and 33 s. The treatment consisted of one cycle of Fab‐IR700 administration followed by NIR light irradiation, and the mice were subjected to two or four treatment cycles.

2.9. Flow Cytometric Analysis

The tumors were harvested and digested with Collagenase I and DNase I (FUJIFLIM Wako Pure Chemical Corporation, Osaka, Japan). The cell suspensions were passed through 40‐μm filters, and the single cells thus obtained were suspended in PBS supplemented with 2% FBS and 1 mM EDTA. Tumor cells were hemolyzed and incubated with anti‐CD16/32 2.4G2 (BD Biosciences, San Jose, CA, USA) to reduce FcγR binding. All samples were stained with antibodies specific for CD45 (BioLegend, clone 30‐F11), CD4 (BioLegend, clone GK1.5), CD8 (BioLegend, San Diego, CA, USA, clone 53‐6.7), IA/IE (BD Bioscience, clone M5/114.15.2), CD11c (BD Bioscience, clone HL3), CD11b (BioLegend, clone M1/70), and F4/80 (BioLegend, clone BM8). The stained samples were analyzed by Guava easyCyte single flow cytometer with guavaSoft 3.3 software (Cytek Biosciences).

2.10. Histological Analysis

Tumors were resected 24 h postirradiation, embedded in optimal cutting temperature compound, and frozen. Ten‐micrometer‐thick frozen sections were cut and mounted onto glass slides. Hematoxylin‐eosin (HE) staining was performed as per standard protocols. For immunohistochemical staining, the sections were incubated with 3% H2O2 to block the endogenous peroxidase activity, followed by incubation with Blocking One Histo (Nacalai Tesque) to prevent nonspecific binding. The sections were probed with rabbit anti‐CD206 antibody (1:500 diluted, Abcam, Cambridge, MA, USA), followed by peroxidase‐conjugated goat anti‐rabbit immunoglobulins (Agilent Technologies, Santa Clara, CA, USA). The Peroxidase Stain DAB Kit (Brown Stain) (Nacalai Tesque) was used to develop antibody staining, and counterstaining was performed using hematoxylin.

2.11. Statistical Analysis

Data have been expressed as the mean ± standard error of the mean (SEM). Statistical analyses were performed using JMP Pro 17.0.0 software (SAS Institute Inc., Cary, NC, USA). Differences in mean values were assessed using the Student's t‐test. Tukey–Kramer test was used. The survival curves were compared using the log‐rank test. Differences were considered statistically significant when p values were < 0.05.

3. Results

3.1. Selective Cytotoxicity of αCSF1R‐ IR700 in In Vitro Coculture Settings

To evaluate whether Fab‐IR700 selectively targets macrophages, we conducted in vitro coculture experiments using MC38 cancer cells and RAW264 macrophages. First, MC38 cells cultured alone were incubated with Fab‐IR700 and subsequently observed under a fluorescence microscope. No IR700 fluorescence was detected, indicating that Fab‐IR700 does not bind to MC38 cells. To distinguish MC38 cells in fluorescence images, MC38 cells were labeled with a CellTrace reagent, and the green fluorescence was observed under the microscope (Figure 1A). In contrast, RAW264 cells cultured alone and incubated with Fab‐IR700 exhibited IR700 fluorescence (Figure 1B). These results demonstrate that MC38 and RAW264 cells can be distinguished based on CellTrace and IR700 fluorescence, respectively. Next, coculture experiments of MC38 and RAW264 cells were performed. After NIR light irradiation, only RAW264 cells that show IR700 fluorescence were stained with the dead cell indicator EthD‐1, whereas MC38 cells remained viable (Figure 1C). These results indicate that Fab‐IR700‐mediated NIR‐PIT selectively induces cytotoxicity in CSF1R‐expressing cells.

FIGURE 1.

FIGURE 1

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In vitro microscopic observation of NIR‐PIT (A–C) and in vivo pharmacokinetics of Fab‐IR700 (D, E). (A) MC38 cancer cells cultured alone were labeled with a CellTrace reagent. (B) RAW264 macrophages cultured alone with Fab‐IR700 exhibited IR700 fluorescence. (C) co‐culture experiments of MC38 and RAW264. After light irradiation, only RAW264 cells that show IR700 fluorescence were stained with the dead cell indicator EthD‐1. Scale bar: 20 μm. (D) In vivo NIR fluorescence images of tumor‐bearing mice intravenously injected with Fab‐IR700. (E) Changes in fluorescence intensity in the tumor and background. Data are the mean ± SEM (n = 5). Statistical significance was determined by Student's t‐test (*p < 0.05).

3.2. Pharmacokinetics of αCSF1R‐ IR700 in Tumor‐Bearing Mice

To investigate the in vivo pharmacokinetics of Fab‐IR700, the conjugate was intravenously administered to MC38 tumor‐bearing mice, and fluorescence imaging was performed at 0.5–72 h postinjection. The fluorescence images of the dorsal and ventral sides of the mice are shown in Figure 1D, and the quantified values of the tumor and background signals are shown in Figure 1E. The results indicated maximum tumor accumulation and a statistically significant difference at 6 h postinjection, based on which we performed NIR light irradiation 6 h after Fab‐IR700 injection in the subsequent experiments.

3.3. Therapeutic Effects of Two Cycles of NIR‐ PIT Targeting TAMs

The TAMs‐targeted NIR‐PIT regimen is shown in Figure 2A. The mice were subjected to two cycles of drug administration and NIR light exposure at the dose of 50 J/cm2. The cytotoxicity of NIR‐PIT utilizing antibody‐IR700 conjugates is driven by a photoinduced ligand‐release reaction, which is unique to IR700 [13, 16]. Upon NIR light irradiation, IR700 undergoes a dramatic change in physical properties from hydrophilic to hydrophobic due to the release of water‐soluble axial ligands. This alteration leads to the formation of water‐insoluble aggregates of antibody‐IR700 on the plasma membrane of target cells, resulting in membrane rupture. While innate IR700 is fluorescent, the aggregated IR700 molecules do not emit fluorescence. Therefore, the decrease in IR700 fluorescence can be used to monitor aggregate formation in tumors. As shown in Figure 2B, the intra‐tumoral fluorescence signals of IR700 disappeared after NIR light irradiation. This was accompanied by a significant reduction in the tumor‐to‐background ratio of IR700 fluorescence postirradiation (Figure 2C). These changes were indicative of the aggregation and cytotoxicity of Fab‐IR700 against the TAMs. Consistent with this, NIR‐PIT significantly decreased tumor volume compared to that in the untreated and light‐treated control groups (Figure 2D–F; Control vs. NIR‐PIT p = 0.0032, Light alone vs. NIR‐PIT p = 0.0028, day 21). Furthermore, the survival rate in the NIR‐PIT group was higher compared to that in the control, light alone, and Fab‐IR700 alone groups (Figure 2G). Taken together, these results suggest that two cycles of NIR‐PIT with αCSF1R Fab‐IR700 can effectively inhibit tumor growth in mice.

FIGURE 2.

FIGURE 2

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Therapeutic effects of 2‐cycle TAMs‐targeted NIR‐PIT. (A) Schematic representation of the treatment regimen: One cycle of Fab‐IR700 administration followed by NIR light irradiation to the tumor. A total of two cycles were performed. (B) In vivo fluorescence images before and after NIR light irradiation. IR700 fluorescence signals in the tumor disappeared after NIR irradiation. (C) Tumor‐to‐background ratio of IR700 fluorescence. Data are the mean ± SEM (n = 5). Statistical significance was determined by Student's t‐test (*p < 0.01). (D) Spider plot showing tumor volume in each mouse from the indicated groups. (E) Changes in mean tumor volume. (F) Mean tumor volume on day 21. Data are the mean ± SEM (n = 4–5). Statistical significance was determined by Tukey–Kramer test (*p < 0.01). (G) Kaplan–Meier survival curves. Statistical significance between control and NIR‐PIT was determined by Log‐rank test (*p < 0.05).

3.4. Therapeutic Effects of Four Cycles of NIR‐ PIT Targeting TAMs

We extended the NIR‐PIT regimen to four cycles to enhance its therapeutic effects (Figure 3A). As shown in Figure 3B,C, 4‐cycle NIR‐PIT achieved greater tumor suppression in terms of the mean tumor volume. This was accompanied by a significant improvement in the survival rates of the tumor‐bearing mice posttreatment (Figure 3D). These findings indicate that CSF1R‐targeted NIR‐PIT is an effective anticancer strategy.

FIGURE 3.

FIGURE 3

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Therapeutic effects of 4‐cycle TAMs‐targeted NIR‐PIT. (A) Schematic representation of the treatment regimen. (B) Spider plot of tumor volume in each mouse from the control and NIR‐PIT groups. The same control group was used for the experiments shown in Figures 2 and 3. (C) Changes in mean tumor volume. Data are the mean ± SEM (n = 4). Statistical significance was determined by Student's t‐test (*p < 0.05, **p < 0.01). (D) Kaplan–Meier survival curves. Statistical significance between control and NIR‐PIT was determined by Log‐rank test (*p < 0.05).

3.5. Photo‐Induced Elimination of TAMs in Tumors Treated With NIR‐ PIT

To evaluate histological changes induced by NIR‐PIT, the tumors were harvested 1 day after NIR light exposure and analyzed by HE staining. As shown in Figure 4A, there were extensive necrotic areas in the tumor tissues subjected to NIR‐PIT, and the characteristics were the same as that observed for cancer‐targeted NIR‐PIT [17, 18, 19]. The depletion of TAMs was assessed through immunostaining and flow cytometry. As shown in Figure 4B,C, there were fewer TAMs and M2‐like macrophages in the tumors treated with NIR‐PIT compared to the control tumors. Some tumors in the control group on day 8 showed a lower ratio of TAMs and M2‐like macrophages compared with the NIR‐PIT group, and the variability observed in TAM may reflect interindividual heterogeneity in the tumor microenvironment. Thus, CSF1R‐targeted NIR‐PIT using Fab‐IR700 can deplete TAMs in light‐irradiated tumors and exhibit therapeutic outcomes.

FIGURE 4.

FIGURE 4

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Histological validation of TAMs‐targeted NIR‐PIT with Fab‐IR700. (A) HE staining images taken on 1 day after NIR light exposure in a single cycle of NIR‐PIT performed on day 7. Intensive necrotic areas surrounded by yellow dot lines were observed in NIR‐PIT‐treated tumors. Scale bar: 200 μm (10×), 50 μm (40×). (B) CD206 IHC images taken on day 1 after NIR light exposure. Scale bar: 50 μm. (C) Quantitative evaluation of TAMs (F4/80+ CD11b+) and M2‐like macrophage (CD206+ F4/80+) on 1 day and 5 days after NIR light exposure on day 8 and 12, respectively. The number of TAMs decreased in tumors treated with NIR‐PIT compared to the control tumors. Data are the mean ± SEM (n = 3–5). Statistical significance was determined by Student's t‐test (*p < 0.05, **p < 0.01).

3.6. NIR‐PIT Established Durable Antitumor Immunity

Immunological memory refers to the ability of the immune system to recognize previously encountered antigens, and is primarily mediated by memory T cells and B cells [20, 21, 22]. The presence of tumor‐specific memory is crucial to prevent relapse due to residual tumor cells. To explore the possibility of long‐term immune protection, MC38 tumor‐bearing mice were treated with four cycles of TAMs‐targeted NIR‐PIT (Figure 5A). Four out of twenty five mice demonstrated a complete response. On day 43 postinoculation, the mice were reinjected with MC38 cells into the contralateral leg (Figure 5A). Tumor recurrence was not observed in mice that achieved complete response, whereas tumors grew unabated after inoculation of cancer cells in the control mice (Figure 5B). Consistent with this, the induction of a sustained antitumor effect after TAMs‐targeted NIR‐PIT extended the survival of mice after tumor rechallenge (Figure 5C). These results suggest that NIR‐PIT with anti‐CSF1R Fab‐IR700 can contribute to a durable antitumor immune response and prevent tumor recurrence.

FIGURE 5.

FIGURE 5

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Tumor rechallenge in mice with complete response after 4‐cycle NIR‐PIT. (A) Schematic representation of the treatment regimen. MC38 cells were reinoculated contralaterally on day 43 after the primary inoculation. (B) Changes in mean tumor volume (n = 4 in NIR‐PIT group and n = 4 in control group). (C) Kaplan–Meier survival curves (n = 4 in NIR‐PIT group and n = 4 in control group). Statistical significance between control and NIR‐PIT was determined by Log‐rank test (*p < 0.05).

3.7. TAMs‐Targeted NIR‐PIT Altered the Intra‐Tumoral Immune Cell Populations

To assess the changes in the immune microenvironment induced by NIR‐PIT, we analyzed the intra‐tumoral immune cell populations at different time points after the 1‐ or 2‐cycle therapy. As shown in Figure 6A, the percentage of macrophages, dendritic cells (DCs), and CD45+CD4+ cells decreased significantly on 5 days following 1‐cycle NIR‐PIT compared to that in control tumors (day 12). On day 14, CD45+CD8+ cells increased significantly in the NIR‐PIT group, suggesting enhanced anticancer immune responses. In contrast, the decrease in DC and CD45+CD4+ cells was not observed on 5 days and 7 days after 2‐cycle NIR‐PIT (day 16 and 18) (Figure 6B). Especially, the percentage of DCs increased significantly in NIR‐PIT‐treated tumors compared to that in control tumors. In this study, CD4+ and CD8+ cells were identified based on CD45 expression according to previous literatures [23, 24]; however, the lack of CD3 staining may limit the precise identification of T cell populations because CD4+/CD8+ monocytes/macrophages and NK cells could be included. These results indicate that the immune cell populations in tumors dynamically change after treatments.

FIGURE 6.

FIGURE 6

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Percentage of dendritic cells (DCs) (IA/IE+ CD11+), CD45+CD4+ cells, and CD45+CD8+ cells in tumors after TAMs‐targeted NIR‐PIT. Analysis was conducted on 5 days and 7 days after 1‐cycle NIR‐PIT (day 12 and 14, respectively) (A) and 5 days and 7 days after 2‐cycle NIR‐PIT (day 16 and 18, respectively) (B). Data are presented as the mean ± SEM (n = 3). Statistical significance was determined by Student's t‐test (*p < 0.05).

4. Discussion

Tumor‐targeted NIR‐PIT with cetuximab‐IR700 conjugate (cetuximab sarotalocan sodium) has been approved for the treatment of inoperable head and neck cancer patients in Japan, and a world‐wide phase III clinical trial is ongoing. In addition, cetuximab‐IR700 has been clinically tested for other epidermal growth factor receptor (EGFR)‐expressing cancers such as esophageal cancer (jRCT2080224666), stomach cancer (jRCT2080224884), and vulvar/cervical cancer (jRCT2011240034). The previous data demonstrate that the cancer‐cell‐targeted NIR‐PIT would be an effective treatment [25]. In the present study, NIR‐PIT using antibody‐IR700 conjugates was applied for photoinduced elimination of CSF1R‐expressing cells in solid tumors (Figure 7). Currently, four cycles of NIR‐PIT are performed in clinical situations. We found that the 4‐cycle NIR‐PIT regimen significantly suppressed tumor growth and prolonged survival rates, suggesting that it is an effective method for cancer control (Figure 3). We also evaluated the efficacy of the shorter 2‐cycle regimen and found that the therapeutic effects of fewer cycles of NIR‐PIT were less potent. Nonetheless, significant tumor suppression was observed with 2‐cycle NIR‐PIT when a higher light dose of 100 J/cm2 was used, indicating that an adequate irradiation dose can facilitate NIR‐PIT against TAMs (Figure S1). The recurrence periods after the final treatment were comparable between the 2‐cycle and 4‐cycle regimens, suggesting that TAM depletion occurs during the early treatment cycles. In other words, most TAMs may be eliminated after the 2‐cycle, whereas additional treatment cycles may suppress their re‐accumulation during the treatment period, thereby maintaining an antitumor microenvironment. A significant increase in AST levels was observed in the treated mice (Figure S2A), which is consistent with previous reports of cancer cell‐targeted NIR‐PIT and is considered to reflect systemic responses to rapid cell death rather than direct hepatotoxicity [26]. In contrast, creatinine levels remained near the detection limit across all groups, suggesting minimal impact on renal function (Figure S2B). From these results, the TAMs‐targeted NIR‐PIT with anti‐CSF1R Fab‐IR700 conjugates and light irradiation would be a new strategy to handle malignant tumors, although comprehensive assessment of the safety profile would be important for further development of CSF1R‐targeted NIR‐PIT.

FIGURE 7.

FIGURE 7

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A scheme explaining the photo‐induced elimination of TAMs using near‐infrared photoimmunotherapy (NIR‐PIT).

While NIR‐PIT successfully eliminated the TAMs (Figure 4B,C), we did not observe an increase in the proportion of antitumor immune cells, such as DCs, CD45+CD4+ cells, and CD45+CD8+ cells, 5 days after 1‐cycle NIR‐PIT (Figure 6A), which might be related to the nonspecific effects by reactive oxygen species generated from IR700 [27, 28, 29]. Nevertheless, TAMs‐targeted NIR‐PIT altered the intra‐tumor immune landscape over a period of few days. These changes may be better elucidated though long‐term and noninvasive approaches such as positron emission tomography (PET) [30, 31]. PET is a molecular imaging technique that utilizes radiolabeled pharmaceuticals to specifically target and can visualize immune‐related molecules in vivo, thus enabling real‐time and whole‐body monitoring of treatment‐induced changes. Notably, the inhibition of tumor recurrence in the NIR‐PIT‐treated mice upon rechallenge is indicative of the establishment of adaptive immunity (Figure 5). Antibodies targeting immune checkpoints such as programed cell death‐1 (PD‐1) and its ligand (PD‐L1), and cytotoxic T‐lymphocyte antigen‐4 (CTLA4), can modulate the immunosuppressive TME and enhance antitumor effects [32]. Therefore, the combination of TAMs‐targeted NIR‐PIT and immune checkpoint inhibitors may act synergistically against “cold” tumors, which are characterized by a low immune infiltration and poor response to immunotherapy. We will explore this therapeutic approach in different murine cancer models in future studies.

Long‐term tumor control can be achieved once antitumor immune responses are activated. In our study, a complete response was observed in 16% (4/25) of the mice in the 4‐cycle NIR‐PIT group with 50 J/cm2 (Figure 5) and in 33% (2/6) of the mice in the 2‐cycle NIR‐PIT group with 100 J/cm2 (Figure S1). The variability in treatment outcomes may reflect differences in immune activation among individual mice. To achieve complete responses even in mice with weaker immune activation and to improve the overall CR rate, it will be important to optimize treatment parameters such as irradiation intensity and the number of treatment cycles. Tumor growth was not observed upon rechallenge in any of the mice that exhibited a complete response in the 4‐cycle NIR‐PIT group with 50 J/cm2, suggesting that elimination of TAMs by NIR‐PIT induced long‐lasting antitumor immunity. Although we could not analyze memory T cells due to the lack of definite markers [33, 34], the increase in DCs, the key antigen‐presenting cells involved in antitumor adaptive immunity, is consistent with the activation of antitumor immune responses and the subsequent induction of memory immune responses following TAM‐targeted NIR‐PIT (Figure 6B). Nagaya et al. have previously shown that cancer cell‐targeted NIR‐PIT using antibody‐IR700 conjugate can elicit a long‐term antitumor immune response through immunogenic death of cancer cells [35]. However, cancer cell‐targeted NIR‐PIT is not a feasible option for tumors lacking specific antigens. On the other hand, TAMs‐targeted NIR‐PIT has the potential to establish durable immune responses against such tumors.

TAMs are the most abundant immunosuppressive cells within the TME, [36] followed by regulatory T cells (Tregs), myeloid‐derived suppressor cells (MDSCs), and cancer‐associated fibroblasts (CAFs), which contribute significantly to cancer progression [37, 38]. A clinical trial of Tregs‐targeted NIR‐PIT with anti‐CD25 antibody‐IR700 conjugates has been initiated in Japan (jRCT2031220721). In addition, previous reports have demonstrated the elimination of MDSCs and CAFs by NIR‐PIT, suggesting the versatility of this approach [39, 40]. Zhang et al. showed that CD206‐targeted NIR‐PIT using an anti‐CD206 antibody‐IR700 conjugate effectively suppressed tumor growth in a murine model by depleting M2‐like TAMs [41]. Although CD206 is an established marker of M2 macrophages, the M2d macrophage subpopulation, which is abundant in the TME, expresses relatively low levels of CD206 [42]. Therefore, CSF1R has recently gained attention as a more broadly applicable marker for targeting TAMs. Our findings have demonstrated that CSF1R‐expressing TAMs are an attractive target for NIR‐PIT. Nevertheless, due to the heterogeneity of the TME among individual patients, it is challenging to select the most appropriate immunosuppressive cell type for NIR‐PIT to achieve optimal therapeutic efficacy. Therefore, it would be beneficial to expand the application of NIR‐PIT to multiple immunosuppressive cell types to treat diverse cancers. Moreover, different immunosuppressive populations act synergistically to aid tumor progression. As a result, multiple antibody‐IR700 conjugates targeting these cell types may be a suitable option for treating refractory cancers.

In conclusion, NIR‐PIT with anti‐CSF1R Fab‐IR700 conjugates successfully eliminated TAMs and enhanced antitumor immunity to prevent recurrence. Although the safety of this approach will have to be confirmed for clinical translation, TAMs‐targeted NIR‐PIT is a promising new strategy to treat recalcitrant tumors.

Author Contributions

Kohei Nakajima: conceptualization, funding acquisition, investigation, visualization, writing – original draft. Tomomi Uchiyama: investigation, visualization, writing – original draft. Kazuki Funayama: investigation. Yuto Goto: investigation. Akari Mukaimine: investigation. Naoya Ieda: investigation. Mikako Ogawa: funding acquisition, resources, supervision, writing – review and editing.

Funding

This work was supported by JSPS KAKENHI Grant Number 19H03593, 21K20706, and 22H03009; Photo‐excitonix Project in Hokkaido University; JSPS J‐PEAKS Grant Number JPJ00420230001.

Ethics Statement

Animal studies: Studies were conducted according to the Guidelines for the Care and Use of Laboratory Animals and approved by the Hokkaido University Animal Care Committee (#20‐0056).

Conflicts of Interest

Dr. Ogawa has received research grants from Rakuten Medical Inc. The funder was not involved in the study design, analysis, and interpretation of the data. Other authors do not have conflicts of interest.

Supporting information

Figure S1: Therapeutic effects of 2‐cycle TAMs‐targeted NIR‐PIT at light dose of 100 J/cm2. (A) Schematic representation of the treatment regimen. (B) Spider plot showing tumor volume in each mouse from the indicated groups. (C) Mean tumor volume on day 21. Data are the mean ± SEM (n = 4–5). (D) Kaplan–Meier survival curves.

Figure S2: Plasma AST and blood creatinine at 24 h after 1‐cycle NIR‐PIT to evaluate systemic toxicity. (A) The values of AST were determined with LabAssay AST kit (FUJIFLIM Wako Pure Chemical Corporation) in a standard protocol. Data are the mean ± SEM (n = 6–8). Statistical significance was determined by Dunnett's t‐test (*p < 0.05). (B) The values of creatinine were evaluated by i‐STAT analyzer (Princeton, NJ, USA). The detection limit of this methods was 0.2 mg/dL.

CAS-9999-0-s001.pdf (289KB, pdf)

Acknowledgments

The authors acknowledge the generous provision of IR700 NHS ester by Rakuten Medical Inc., which was used for preclinical studies on NIR‐PIT.

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Associated Data

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Supplementary Materials

Figure S1: Therapeutic effects of 2‐cycle TAMs‐targeted NIR‐PIT at light dose of 100 J/cm2. (A) Schematic representation of the treatment regimen. (B) Spider plot showing tumor volume in each mouse from the indicated groups. (C) Mean tumor volume on day 21. Data are the mean ± SEM (n = 4–5). (D) Kaplan–Meier survival curves.

Figure S2: Plasma AST and blood creatinine at 24 h after 1‐cycle NIR‐PIT to evaluate systemic toxicity. (A) The values of AST were determined with LabAssay AST kit (FUJIFLIM Wako Pure Chemical Corporation) in a standard protocol. Data are the mean ± SEM (n = 6–8). Statistical significance was determined by Dunnett's t‐test (*p < 0.05). (B) The values of creatinine were evaluated by i‐STAT analyzer (Princeton, NJ, USA). The detection limit of this methods was 0.2 mg/dL.

CAS-9999-0-s001.pdf (289KB, pdf)

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