Cancer-associated fibroblasts (CAFs) are a heterogenous group of activated fibroblasts and a major component of the tumor stroma. CAFs may be derived from fibroblasts, epithelial cells, endothelial cells, cancer stem cells, adipocytes, pericytes, or stellate cells. These complex origins may underlie their functional diversity, which includes pro-tumorigenic roles in extracellular matrix remodeling, the suppression of anti-tumor immunity, and resistance to cancer therapy. Several methods for targeting CAFs to inhibit tumor progression and enhance anti-tumor immunity have recently been reported. While preclinical studies have shown promise, to date they have been unsuccessful in human clinical trials against melanoma, breast cancer, pancreas cancer, and colorectal cancers. This review summarizes recent and major advances in CAF-targeting therapies, including DNA-based vaccines, anti-CAF CAR-T cells, and modifying and reprogramming CAF functions. The challenges in developing effective anti-CAF treatment are highlighted, which include CAF heterogeneity and plasticity, the lack of specific target markers for CAFs, the limitations in animal models recapitulating the human cancer microenvironment, and the undesirable off-target and systemic side effects. Overcoming these challenges and expanding our understanding of the basic biology of CAFs is necessary for making progress towards safe and effective therapeutic strategies against cancers in human patients.

Cancer-associated fibroblasts (CAFs) constitute a prominent cellular component of the tumor stroma, with various pro-tumorigenic roles. Numerous attempts to target fibroblast activation protein (FAP), a highly expressed marker in immunosuppressive CAFs, have failed to demonstrate anti-tumor efficacy in human clinical trials. Near-infrared photoimmunotherapy (NIR-PIT) is a highly selective tumor therapy that utilizes an antibody-photo-absorbing conjugate activated by near-infrared light. In this study, we examined the therapeutic efficacy of CAF depletion by NIR-PIT in two mouse tumor models. Using CAF-rich syngeneic lung and spontaneous mammary tumors, NIR-PIT against FAP or podoplanin was performed. Anti-FAP NIR-PIT effectively depleted FAP⁺ CAFs, as well as FAP⁺ myeloid cells, and suppressed tumor growth, whereas anti-podoplanin NIR-PIT was ineffective. Interferon-gamma production by CD8 T and natural killer cells was induced within hours after anti-FAP NIR-PIT. Additionally, lung metastases were reduced in the treated spontaneous mammary cancer model. Depletion of FAP⁺ stromal as well as FAP⁺ myeloid cells effectively suppressed tumor growth in bone marrow chimeras, suggesting that the depletion of both cell types in one treatment is an effective therapeutic approach. These findings highlight a promising therapy for selectively eliminating immunosuppressive FAP⁺ cells within the tumor microenvironment.

Metastatic and relapsed osteosarcoma (OS) remains difficult to treat despite advanced surgical techniques, intensified chemotherapy, and targeted therapies. Adoptive immunotherapies such as chimeric antigen receptor (CAR) T cells, are in their nascent stage, but remain a viable therapeutic strategy for patients with aggressive solid tumors such as OS. Folate receptor- (FOLR1) has been functionally implicated in OS pathophysiology, providing rationale as a potential therapeutic target. We recently advanced a FOLR1-specific CAR T cell product (FH FOLR1-CART) into a trial in infant AML (NCT06609928) and now evaluate this CAR construct against OS. We provide comprehensive FOLR1 transcript and protein expression profile in OS patients, cell lines and patient-derived xenografts, substantiating its significance as a therapeutic target. We further evaluate in vitro and in vivo efficacy of FH FOLR1-CART in both standard and patient-derived OS cell lines and xenograft models. FOLR1 transcript is expressed in the overwhelming majority of OS primary patient specimens, in OS cell lines, and patient-derived models. FH FOLR1-CART exhibit robust in vitro activation and potent cytotoxicity against FOLR1-expressing OS cell lines and primary OS patient samples. More importantly, FH FOLR1-CART demonstrates potent anti-tumor activity in both localized and metastatic in vivo cell-derived and patient-derived xenograft models, with complete tumor eradication. These results demonstrate a potential therapeutic option for patients with advanced OS. FH FOLR1 CART is advancing to an early phase trial in relapsed/refractory OS at Fred Hutch Cancer Center and Seattle Children’s Hospital.

Spatial biology has emerged as a transformative field, offering insights into cellular interactions and organization within tissues. The field has evolved rapidly since the coining of the term “spatial omics.” Now, the ability to spatially resolve proteins, RNA, chromatin, and lipids is becoming widespread, and the technologies are continually refined. Reagents to support the analysis of veterinary species are available and more are emerging. These new tools will allow pathologists and scientists to unravel the intricate interplay between tissue architecture and diverse cellular phenotypes. By integrating histological observations with spatially resolved genomic data, spatial biology holds immense potential for advancing diagnostic and therapeutic strategies in veterinary medicine. These tools will undoubtedly equip veterinary pathologists to better decipher complex disease processes and identify novel therapeutic targets.

In vivo functional evaluation of FH FOLR1-CART in a localized U-2 OS xenograft model. A, Average radiance emitted by tumor measured over 180 days from intrafemoral tumor inoculation in mice treated with FH FOLR1-CART vs. NT T cells. B, Kaplan–Meier survival curves of xenografts treated with NT or FH FOLR1-CAR T cells. n = 2 per group. Statistical differences in survival evaluated using Mantel–Cox log-rank test. C, Bioluminescence imaging of localized U-2 OS tumor inoculated intrafemorally in mice treated with NT or FH FOLR1-CAR T cells. Radiance scale indicates progressive osteosarcoma from blue to red. *Day 13 images use a minimum radiance cutoff of 1e6 p/second/cm2/sr to show the presence of tumor engraftment prior to treatment. Minimum radiance cutoff of 2.5e6 p/second/cm2/sr used for posttreatment images to avoid noise created by excess radiance detected. X indicates death. D, day. D, Axial T2-weighted fat-saturated images of mouse femur treated with NT T cells at 84 days after local osteosarcoma cell line inoculation. Note the T2 hyperintense lobulated soft tissue mass (solid white arrow) involving the distal right femur. The bladder (star), noted more medially, also demonstrates T2 hyperintensity. E, The bone underlying the mass at 84 days after tumor cell line injection demonstrates abnormal morphology with diffuse marrow signal change and cortical destruction (open arrow). F, Axial T2-weighted fat-saturated images of mouse femur treated with FH FOLR1-CAR T cells at 84 days after tumor inoculation. Note the absence of any soft tissue mass or bone marrow signal changes of the distal right femur (solid arrows).

FH FOLR1-CART antitumor activity against U-2 OS osteosarcoma cell line in vitro. A, FOLR1 expression detected by flow cytometry by U-2 OS, WSU-AML (standard M7, FOLR1-positive AML), and Kasumi (standard FOLR-negative AML). Red, tumor stained with PE-labeled anti-FOLR1; unfilled, isotype control. B, Increased FH FOLR1-CAR T cell–induced tumor cell lysis detected by flow cytometry after co-culture at 6 and 24 hours at various E:T ratios, compared with tumor cell lysis induced by NT T cells. C, Representative images from the IncuCyte imaging platform at hours 0 and 20.5 of co-culture. Green, live U-2 OS tumor cells; red, dead cells. D, Concentration (pg/mL) of secreted IL-2, IFN-γ, and TNFα detected in co-culture supernatant following 24 hours of 1:1 CD8+ T cell-to-U-2 OS ratio. *, P < 0.0005.