Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Apr 18;22(1):367.
doi: 10.1186/s12967-024-05174-y.

Mesothelin-based CAR-T cells exhibit potent antitumor activity against ovarian cancer

Jing Guo  1 , Xiaozhu Zeng  1 , Yongjie Zhu  1 , Dong Yang  1 , Xudong Zhao  2
Affiliations

Mesothelin-based CAR-T cells exhibit potent antitumor activity against ovarian cancer

Jing Guo et al. J Transl Med. .

Abstract

Background: Ovarian cancer (OC) is characterized by its rapid growth and spread which, accompanied by a low 5-year survival rate, necessitates the development of improved treatments. In ovarian cancer, the selective overexpression of Mucin-16 (MUC16, CA125) in tumor cells highlights its potential as a promising target for developing anti-tumor therapies. However, the potential effectiveness of CAR-T cell therapy that targets MUC16 in ovarian cancer cells is unknown.

Methods: The expression of MUC16 in viable OC cells was detected using immunofluorescence and flow cytometry techniques. A MSLN-CAR construct, comprising the MUC16-binding polypeptide region of mesothelin (MSLN), a CD8 hinge spacer and transmembrane domain, 4-1BB, and CD3ζ endo-domains; was synthesized and introduced into T cells using lentiviral particles. The cytotoxicity of the resultant CAR-T cells was evaluated in vitro using luciferase assays. Cytokine release by CAR-T cells was measured using enzyme-linked immunosorbent assays. The anti-tumor efficacy of the CAR-T cells was subsequently assessed in mice through both systemic and local administration protocols.

Results: MSLN-CAR T cells exhibited potent cytotoxicity towards OVCAR3 cells and their stem-like cells that express high levels of MUC16. Also, MSLN-CAR T cells were inefficient at killing SKOV3 cells that express low levels of MUC16, but were potently cytotoxic to such cells overexpressing MUC16. Moreover, MSLN-CAR T cells delivered via tail vein or peritoneal injection could shrink OVCAR3 xenograft tumors in vivo, with sustained remission observed following peritoneal delivery of MSLN-CAR T cells.

Conclusions: Collectively, these results suggested that MSLN-CAR T cells could potently eliminate MUC16- positive ovarian cancer tumor cells both in vitro and in vivo, thereby providing a promising therapeutic intervention for MUC16-positive patients.

Keywords: CAR-T cells; MUC16 (CA125); Mesothelin; Ovarian cancer.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no financial or non-financial competing interests.

Figures

Fig. 1
Fig. 1
MSLN-CAR T cells can efficiently lyse MUC16-positive human ovarian cancer cells. A Schematic illustration of a CD19-CAR and a MSLN-CAR T vector, respectively. B, C Quantitative analysis of MUC16 protein expression levels in ovarian cells (SKOV3, OVCAR3) by Western blotting (B) and flow cytometry (C). Tubulin was used as a loading control. Lane 1 = molecular marker; Lanes 2 = SKOV3 cell lysate; Lanes 3 = OVCAR3 cell lysate; Lanes 4 = MUC16 overexpression in 293T cell lysate. B and C consist of three technical replicates. The pictures represent one example of three technical replicates. D The cytotoxicity of MSLN-CAR T cells on ovarian cancer cell lines was quantified using a luciferase assay. Primary human T cells transduced with the indicated lentiviruses were co-incubated with target cells expressing luciferase at an effector to target (E:T) ratio of 2:1 for 20 h. Three independent experiments were performed. E Quantification of IFN-γ (left) and TNF-α (right) release in response to coculture with CD19-CAR or MSLN-CAR T cells at an E:T ratio of 2:1, as measured by ELISA. Data are presented as the mean ± SD, n = 3. F CAR T cells were co-incubated with target cells expressing luciferase at varying effector to target (E:T) ratios for 20 h. D–F consist of three biologic replicates and three technical replicates. Statistics: two-tailed one-way ANOVA. The results are presented as the mean volume ± SD; *P < 0.05, **P < 0.01, ***P < 0.001 vs CD19-CAR or NTD
Fig. 2
Fig. 2
MSLN-CAR T cells target MUC16-overexpressing cells for cytotoxic killing. A Structure of the pLGNe-MUC16 expression plasmid used for overexpression assays. B The level of MUC16 protein overexpression in SKOV3 cell line (left) was evaluated by Western blot (light), with Tubulin was used as a loading control. Lane 1 = molecular marker; Lanes 2 = SKOV3 cell lysate; Lanes 3 = MUC16 overexpression in SKOV3 cell lysate. B and C consist of three technical replicates. The pictures represent one example of three technical replicates. C Flow cytometry was used to detect MUC16 expression on the surface of viable, SKOV3 cells. D Cytotoxicity of MSLN-CAR T cells following their co-culture with MUC16-overexpressing cells as tested with a luciferase-based assay. CAR T cells were incubated with luciferase-expressing target cells for 20 h at an effector to target (E:T) ratio of 2:1. E, F ELISA-based quantification of IFN-γ (E) and TNF-α (F) released in response to coculture with Mock or MSLN-CAR T cells at an E:T ratio of 2:1. G, H CAR T cells were co-incubated with target cells expressing luciferase at varying effector to target (E:T) ratios for 20 h. D–H consist of three biologic replicates and three technical replicates. Statistics: two-tailed one-way ANOVA. The results are presented as the mean volume ± SD; ns = not significant; *P < 0.05, **P < 0.01 vs CD19-CAR or NTD
Fig. 3
Fig. 3
MSLN-CAR T cells efficiently kill cancer stem-like cells. A, B Quantification of RNA transcript levels for CD133, CD44, CD117 and CD24 in ovarian cells (SKOV3, OVCAR3) and their cancer stem cell derivations (SKOV3-CSC and OVCAR3-CSC). For statistical analysis of two groups an unpaired two sample t-test is used. Data were plotted and are shown as mean ± SD; * P < 0.05, *** P < 0.001. C Quantification of RNA transcripts for MUC16 in ovarian cells (SKOV3, OVCAR3) and their cancer stem cell derivations (SKOV3-CSC and OVCAR3-CSC) were determined by confocal imaging. D, E Both flow cytometry and immunofluorescence staining were used to stain live cells. A–D consist of three technical replicates. The pictures represent one example of three technical replicates. F The cytotoxicity of MSLN-CAR T cells on luciferase-expressing target cells was quantified after incubation for 20 h at an E:T ratio of 2:1. Three independent experiments were performed per condition. G ELISAs were used to detect IFN-γ release by T cells in coculture supernatants. E and F consist of three biologic replicates and three technical replicates. Statistics: two-tailed one-way ANOVA. Data are presented as the mean ± SD, n = 3; ***P < 0.001 vs CD19-CAR or NTD
Fig. 4
Fig. 4
Antitumor effects of MSLN-CAR T cells on ovarian cancer cell xenografts in vivo. A Gating strategy of flow cytometry to discriminate TN, TCM, TEM, and TEMRA cells. B Flow cytometry was used to analyze the percentages of Tcm and TemT cell subsets in various CARs, with results reported as mean ± SD, ns means no significance. C NCG mice were intraperitoneally injected with 5 × 105 OVCAR3 cells and imaged on the fifth day. Next, BLI of tumor progression of representative mice receiving intraperitoneally injected or intravenously (tail vein) injected MSLN-CAR T cells, compared to control (CD19-CAR T cell treated) mice. D Quantification of tumor bioluminescence levels at different time points (n = 4 mice per group). (E) Quantification of body weights of mice receiving receiving intraperitoneally injected or intravenously (tail vein) injected MSLN-CAR T cells or CD19-CAR T cells. The experiments were repeated twice, independently, with similar results
Fig. 5
Fig. 5
Prolonged survival of NCG mice with ovarian cancer cell xenograft tumors following intraperitoneal infusion of MSLN-CAR T cells. A Human ovarian tumor OVCAR3 cells were injected intraperitoneally into mice, following which 5 × 106 of either CD19-CAR T cells or MSLN-CAR T cells were injected intraperitoneally. Following treatment with the respective CAR T cells, tumor burden was monitored at the indicated time points using bioluminescent imaging. B Quantification of tumor bioluminescence levels at different time points (n = 6 mice per group). C Kaplan–Meier survival curve of NCG mice treated i.p. with CD19-CAR or MSLN-CAR T cells. The experiments were repeated twice, independently, with similar results. Statistical analysis by two-tailed log rank test; n.d., not detected; *P < 0.05
Fig. 6
Fig. 6
Specificity and safety of MSLN-CAR T cells. A The cytotoxicity of MSLN-CAR T cells on luciferase-expressing target cells was quantified after incubation for 20 h at an E:T ratio of 2:1. Three independent experiments were performed per condition. Statistics: two-tailed one-way ANOVA. Data are presented as the mean ± SD, n = 3; ***P < 0.001 vs CD19-CAR or NTD. B Quantification of retroviral vector copy number (VCN) in transduced T cells (CAR). Measured VCN per cell of MSLN-CAR T cells derived from three healthy donors above. Cells were assessed 96 h post transduction and the transduction MOI and efficiency were the same as the above. The experiment was conducted using three biologic replicates, each one including three technical replicates. C Levels of IFN-γ (left), and TNF-α (right) were measured by ELISA on serum collected from peripheral blood on three days after immune effector cell infusion. For statistical analysis of two groups an unpaired two sample t-test is used. Data are presented as the mean ± SD, n = 3. **P < 0.01 vs CD19-CAR. D Designated organs were analyzed after fluorescent staining of immune tissues on day 5 after intraperitoneal or intravenous administration of CAR T cells. Representative confocal microscopy images of CD3+ T cells in major organs (heart, liver, spleen, lung, kidney, brain) (n = 3 mice/group). Scale bar = 100 μm. The experiments were repeated twice, independently, with similar results
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Hagimori M, Kato N, Orimoto A, et al. Development of triple-negative breast cancer-targeted liposomes with MUC16 binding peptide ligand in triple-negative breast cancer cells. J Pharm Sci. 2023;112(6):1740–1745. doi: 10.1016/j.xphs.2023.02.025. - DOI - PubMed
    1. Matulonis UA, Sood AK, Fallowfield L, et al. Ovarian cancer. Nat Rev Dis Primers. 2016;2:16061. doi: 10.1038/nrdp.2016.61. - DOI - PMC - PubMed
    1. Marimuthu S, Lakshmanan I, Muniyan S, et al. MUC16 promotes liver metastasis of pancreatic ductal adenocarcinoma by upregulating NRP2-associated cell adhesion. Mol Cancer Res. 2022;20(8):1208–1221. doi: 10.1158/1541-7786.MCR-21-0888. - DOI - PMC - PubMed
    1. Chaudhary S, Appadurai MI, Maurya SK, et al. MUC16 promotes triple-negative breast cancer lung metastasis by modulating RNA-binding protein ELAVL1/HUR. Breast Cancer Res. 2023;25(1):25. doi: 10.1186/s13058-023-01630-7. - DOI - PMC - PubMed
    1. Felder M, Kapur A, Gonzalez-Bosquet J, et al. MUC16 (CA125): tumor biomarker to cancer therapy, a work in progress. Mol Cancer. 2014;13:129. doi: 10.1186/1476-4598-13-129. - DOI - PMC - PubMed
-