FAP: A stromal target in tumor microenvironment modulation and therapeutic intervention

• Gene Information: Fibroblast activation protein alpha (FAP) is a type II transmembrane serine protease with dipeptidyl peptidase and endopeptidase activity. FAP is encoded by the FAP gene and is closely related to the dipeptidyl peptidase family.
• Protein Expression: FAP is minimally expressed in most normal adult tissues but is highly expressed by activated fibroblasts, especially cancer-associated fibroblasts (CAFs), in many epithelial tumors. FAP expression is also associated with tissue remodeling, wound healing, fibrosis, and chronic inflammation.
• Signaling Pathway/TME Modulation: FAP regulates the tumor microenvironment mainly through protease-dependent extracellular matrix remodeling and stromal-cell modulation. FAP-positive cancer-associated fibroblasts are associated with TGF-β signaling, CXCL12/CXCR4-mediated immune exclusion, VEGF-related angiogenesis, and immunosuppressive cell recruitment. These pathways contribute to tumor progression, impaired T-cell infiltration, and resistance to antitumor immunity.
• Therapeutic Inhibition: FAP-targeted therapeutic strategies include FAP enzyme inhibitors, FAP-targeted antibodies, bispecific antibodies, CAR-T cells, antibody-drug conjugates, immunotoxins, and radioligand therapies. B-hFAP mice provide an in vivo model for evaluating human FAP-targeted therapies in a physiologically relevant tumor microenvironment.

FAP

• Exons 3–26 of the mouse Fap gene encoding the extracellular domain were replaced with human FAP exons 3–26 in B-hFAP mice.
• The genomic region of mouse Fap gene that encodes transmembrane domain and cytoplasmic portion is retained. The promoter, 5’UTR and 3’UTR region of the mouse gene are also retained. The chimeric FAP expression is driven by endogenous mouse Fap promoter, while mouse Fap gene transcription and translation will be disrupted.

• Mouse Fap mRNA was detectable in kidney tissue of wild-type C57BL/6 mice.
• Human FAP mRNA was detectable in homozygous B-hFAP mice and B-CAG-hFAP MC38 cell line, but not in wild-type mice.
• Mouse Fap mRNA and human FAP mRNA were not detectable in homozygous B-Fap KO mice.

Strain specific analysis of FAP gene expression in wild-type C57BL/6 mice and B-hFAP mice by RT-PCR. Murine colon cancer MC38 cells (5×10⁵) and melanoma B16-F10 (2×10⁵) were specifically subcutaneously implanted into wild-type C57BL/6 mice, homozygous B-hFAP mice and homozygous B-Fap KO mice. B-hFAP MC38 cell line which over-expressing human FAP in MC38 cell line was used as a positive control (G7). The mRNA was prepared from kidney tissue of the mice (G1-G6).

• B-hFAP mice were able to establish tumors in vivo.

Subcutaneous homograft tumor growth of MC38 and B16-F10 cells. MC38 cells (5×10⁵) were subcutaneously implanted into B-hFAP mice (female, 7 weeks old n=2). B16-F10 cells (2×10⁵) were subcutaneously implanted into B-hFAP mice (female, 7 weeks old, n=1). Tumor volume and body weight were measured twice a week. (A) Average tumor volume ± SEM. (B)  Body weight (Mean± SEM). Volume was expressed in mm³ using the formula: V=0.5 × long diameter × short diameter².

• Human FAP was exclusively detectable in CD45- cells of tumor tissue from homozygous B-hFAP mice but not in the cells from wild-type C57BL/6 mice or B-Fap KO mice.

Strain specific analysis of FAP expression in wild-type C57BL/6 mice, homozygous B-hFAP mice and B-Fap KO mice by FACS. Murine melanoma B16-F10 (2×10⁵) were specifically subcutaneously implanted into wild-type C57BL/6 mice, homozygous B-hFAP mice and homozygous B-Fap KO mice. Tumor tissue was collected from the three strains of mice, and analyzed by flow cytometry with species-specific anti-hFAP antibody (R&D, FAB3715P-100).

• B-hFAP mice were able to establish tumors in vivo.

Subcutaneous homograft tumor growth of Pan02 cells. Pan02 cells (2×10⁷) were subcutaneously implanted into B-hFAP mice (female, n=3). Tumor volume and body weight were measured twice a week. (A) Average tumor volume ± SEM. (B)  Body weight (Mean± SEM). Volume was expressed in mm3 using the formula: V=0.5 × long diameter × short diameter².

• Human FAP was exclusively detectable in CD45- cells of tumor tissue from homozygous B-hFAP mice but not in the cells from wild-type C57BL/6 mice.

Strain specific analysis of FAP expression in wild-type C57BL/6 mice and homozygous B-hFAP mice by FACS. Murine Pan02 cells (2x10⁷) were specifically subcutaneously implanted into wild-type C57BL/6 mice and homozygous B-hFAP mice. Tumor tissue was collected from the three strains of mice, and analyzed by flow cytometry with species-specific anti-hFAP antibody (R&D, FAB3715P-100).

• Human FAP was was detectable in MEF cells of homozygous B-hFAP mice.

Strain specific FAP expression analysis in homozygous B-hFAP mice by flow cytometry. MEF cells was collected from wild-type C57BL/6 mice (+/+) and homozygous B-hFAP mice (H/H), and protein expression was analyzed with anti-human FAP antibody (R&D, FAB3715P-100) by flow cytometry.

• Mouse sFAP was only detectable in wild-type C57BL/6 mice.
• Human sFAP was exclusively detectable in homozygous B-hFAP mice and human plasma.

Strain specific soluble FAP (sFAP) expression analysis in wild-type C57BL/6 mice and homozygous B-hFAP mice by ELISA. Plasma was collected from wild-type C57BL/6 mice and homozygous B-hFAP mice (female, 8-week-old, n=3). Human plasma was used as positive control. Expression level of mouse and human sFAP were analyzed by ELISA (anti-mouse FAP ELISA kit: abcam, 289903; anti-human FAP ELISA kit: abcam, 256404). Values are expressed as mean ± SEM.

• FAP/Fap mRNA was detected in multiple tissues, with relatively higher levels in lung, adipose, submandibular glad and uterus.

Relative FAP/Fap mRNA expression in different tissues of wild-type C57BL/6 mice and homozygous B-hFAP mice by RT-qPCR. Tissues were collected from wild-type C57BL/6 mice and homozygous B-hFAP mice (female, 8 weeks old, n=3). Relative FAP/Fap mRNA expression levels were normalized to the level in the lung of C57BL/6 mice. The qPCR primers were designed to target an identical sequence shared by human FAP and mouse Fap, with the same amplicon length generated from both templates. Accordingly, the assay detects total FAP/Fap transcripts but does not differentiate human FAP from mouse Fap transcripts. Values are expressed as mean ± SEM.

• Humanized FAP protein was detected in multiple tissues of B-hFAP mice. Weak or tissue-specific signals in wild-type tissues may reflect endogenous mouse FAP expression, antibody cross-reactivity, or non-specific binding.

FAP protein expression profile in wild-type C57BL/6 mice and homozygous B-hFAP mice by western blot. Tissues were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hFAP mice (H/H), and analyzed using an anti-FAP antibody (R&D, AF3715). In B-hFAP mice, FAP signals were detected in spleen, heart, ovary, uterus, skeletal muscle, adipose tissue, colon, small intestine, lung, brain, submandibular gland, liver, kidney, and skin.

Experimental schedule for the induction of bleomycin-induced pulmonary fibrosis and in vivo efficacy evaluation of dexamethasone and talabostat mesylate in B-hFAP mice. Bleomycin was administered intranasally to mice on day 0 and day 1 to induce pulmonary fibrosis. Dexamethasone, a corticosteroid positive-control treatment, was administered intraperitoneally according to the indicated dosing schedule, while talabostat mesylate (MCE, HY-13233A) was administered orally from day 7 to day 20. Mice were monitored throughout the study and sacrificed at the endpoint on day 21. BLM, bleomycin; DEX, dexamethasone; HYP, hydroxyproline.

• B-hFAP mice support bleomycin-induced pulmonary fibrosis modeling and can be used for in vivo evaluation of FAP-targeted interventions.

Body weight and survival in a bleomycin-induced pulmonary fibrosis model using B-hFAP mice and C57BL/6 mice. B-hFAP mice and C57BL/6 mice were intranasally challenged with bleomycin on days 0 and 1. B-hFAP mice were treated with PBS or talabostat mesylate, while C57BL/6 mice were treated with PBS, dexamethasone, or talabostat mesylate according to the indicated schedules. (A) Body weight changes. (B) Survival curve. Values are expressed as mean ± SEM.

• Bleomycin increased lung hydroxyproline levels, confirming collagen accumulation and pulmonary fibrosis induction in B-hFAP mice.

Hydroxyproline analysis in the bleomycin-induced pulmonary fibrosis model using B-hFAP mice and C57BL/6 mice. Lung tissues were collected after bleomycin induction and treatment with PBS, dexamethasone, or talabostat mesylate. Hydroxyproline (HYP) content was measured to evaluate collagen deposition and pulmonary fibrosis severity. (A) HYP per lung. (B) HYP normalized to wet lung weight. Bleomycin increased lung HYP levels compared with saline controls, indicating successful fibrosis induction. Values are expressed as mean ± SEM. Significance was determined by one-way ANOVA test. *P < 0.05, **P < 0.01.