MAFB is a biomarker for cancer severity and prognosis, and is a potential cancer immunotherapy target

doi:10.21203/rs.3.rs-763431/v2

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MAFB is a biomarker for cancer severity and prognosis, and is a potential cancer immunotherapy target

https://doi.org/10.21203/rs.3.rs-763431/v2

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published 31 Aug, 2022

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MAFB is a transcription factor specifically expressed in macrophages. Tumor associated macrophages (TAMs) play a key role in the tumor microenvironment (TME) by inducing immunosuppression, angiogenesis, tumor invasion, and metastasis. However, finding a suitable specific biomarker and target for TAMs is challenging. Our previous study¹ suggested that MAFB could be a suitable marker for tumor-associated macrophages (TAMs) besides MAFB is expressed in anti-inflammatory alternatively activated M2 macrophages in vitro. In the current study, in a cohort of patients with lung adenocarcinoma (n = 120), increased MAFB expression was related to increased metastasis and poor overall survival rate. Our findings indicate that MAFB can be used as a prognostic marker for assessing metastatic potential in patients with lung adenocarcinoma. Further, we showed that MAFB expression was positively correlated with the expression of CD204 and CD68 in hepatocarcinoma, colon and pancreatic cancers. We demonstrated that MAFB could be used as a biomarker for TAMs and consequently, for assessing severity in various human cancers, including lung, liver, colon, and pancreatic cancers, according to the immunohistochemical analysis of the expression of MAFB, CD68, and CD204. In addition, we showed that MAFB was expressed in TAMs expressing Programmed cell death protein-1 and/or Programmed cell death ligand 1 (TAM PD-1⁺ and TAM PD-L1⁺) cells in lung adenocarcinoma and Lewis lung carcinoma (LLC) mouse model. These findings indicate that MAFB can be a potential target for drug development against TAM PD-1⁺ and TAM PD-L1⁺ cells. In summary, transcriptional factor MAFB can be used as a specific biomarker, prognostic marker, and a potential target for cancer immunotherapy against TAMs.

Cancer Biology

Oncology

Nuclear Medicine & Medical Imaging

MAFB

tumor-associated macrophages

biomarker

cancer severity

cancer prognosis

V-maf musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB) is a member of the large Maf transcription factor family² which is expressed in several tissues and is associated with the differentiation of various cell types, including macrophages (Mφs),³ tumor-associated Mφs (TAMs),¹ kidney podocytes,⁴ keratinocytes,^5,6 and pancreatic α-cells and β-cells.^7,8 In the hematopoietic cell lineage, MafB induces monocytes to differentiate into Mφs rather than dendritic cells, suggesting that MafB can shape the Mφ phenotype.^9,10 Moreover, MAFB expressed in Mφs is related to the restoration of homeostasis, which can be achieved by the resolution of inflammation, removal of dead cells,¹¹ and the uptake of oxidized cholesterol.¹² Mutations in human MAFB have been reported to cause diseases, such as multicentric carpotarsal osteolysis and Duane retraction syndrome with focal segmental glomerulosclerosis.^13,14 Furthermore, MAFB is expressed in many disease-specific Mφs, including infiltrating Mφs at the site of bacterial infections,¹⁵ monocyte-derived MHC-II-positive Mφs in myocardial infarction models,¹⁶ atherosclerotic regions,^12,17,18 Mφs in rat hind-limb ischemia models,¹⁹ and kidney-resident Mφs in renal artery stenosis models.²⁰ In addition, MAFB expression has been reported in profibrotic Mφs in lung fibrosis induced by bleomycin,²¹ microglial cells following peripheral nerve injury of the spinal cord,^10,22,23 and peritoneal Mφs in human patients with peritoneal dialysis.²⁴ Moreover, MAFB expression in TAMs has been reported in mouse mammary tumors,²⁵ and human skin, colon, and melanoma tumors.²⁶

In our previous work,¹ we showed that the transcription factor MAFB could be a biomarker for TAMs in both lung adenocarcinoma, and Lewis lung carcinoma (LLC) mouse model; MAFB expression was also observed in anti-inflammatory alternatively activated M2 Mφs in vitro.

The ecosystem of the tumor microenvironment (TME) is characterized by diverse heterogeneity and is rapidly evolving. Homeostasis of the TME is regulated by an intimate crosstalk among its cellular components, involving malignant, endothelial, stromal, and immune cells.²⁷ While the TME comprises both adaptive and innate immune cells, the later can facilitate cancer cell growth and metastasis, induce tumor angiogenesis, suppress antitumor immune response, and modulate response to anticancer therapies. These innate immune cells include Mφs, dendritic cells, neutrophils, myeloid-derived suppressor cells, natural killer cells, and innate lymphoid cells,²⁸ among which monocyte-derived Mφs account for 50% of the tumor mass.²⁹ Mφs can be classified as inflammatory M1, which are classically activated and exert antitumor effect, or immune-suppressive M2, which are alternatively activated and have protumor effect.³⁰ However, Mφs are dynamic cells with functional plasticity.³¹ In fact, M2 Mφs can be broadly classified into M2a, M2b, M2c, and M2d subsets²⁸ based on Mφ polarization inducers as follows: IFNγ and LPS for M1; IL4, IL10, and IL13 for M2a; TLR agonists for M2b; IL10, TNFα, and glucocorticoids for M2c; and TLR and adenosine A2A receptor for M2d.³² Hence, the dynamicity of Mφs accounts for the high degree of heterogeneity of TAMs within the TME and reflects the ability of TAMs to acquire various phenotypic, metabolic, and functional profiles in response to ecosystem fluctuations of the TME³³ among patients suffering from specific cancer types or even within different lesion sites in the same patient.²⁷ Generally, definite TAM subsets (M2) support oncogenesis, angiogenesis, immunosuppression, and therapy resistance, resulting in poor prognosis, whereas other TAM populations (M1) exhibit anti-tumor activity (tumoricidal) and increase the efficacy of various anticancer immunotherapies.³⁴

TAMs are suitable indicators of tumor malignancy³⁵ and multi-marker methods are available to distinguish TAMs.^36–41 There are many diagnostic and prognostic markers for TAMs within the TME. One of the most common ones is the CD68 (pan-Mφ antibody).⁴² The limitation of CD68 application is that it is not singularly expressed by TAMs but is also expressed by many other TME cells, such as malignant epithelial and stromal cells.⁴³ Notably, M1 and M2 subsets cannot be recognized via the single labeling of Mφs with CD68 marker antibodies. Hence, multiplex markers for multiplex Mφs (usually two or three different Mφ markers) have been recently used to identify M1 and M2 subsets but this approach is costly and time consuming.⁴⁴

In addition, CD163 (hemoglobin-scavenger receptor) and CD204 (Mφ-scavenger receptor-1) markers may be used to identify Mφs. CD163 and CD204 have been proposed to affect tumor progression by stimulating metastasis, neovascularization, and immune evasion. They exhibit dual action by inhibiting the antitumor activity of both M1 pro-inflammatory TAM and T-helper (Th1) cells and activating regulatory T cells and Th2 cells.^42,45,46 Although both CD163 and CD204 are expressed specifically on TAM, they have different expression levels, functional significance, and prognostic values in various cancer types. This suggests that CD163 and CD204 are not expressed in identical TAM populations.^36–40 Accordingly, levels of both CD163 and CD204 are monitored to evaluate the polarization of TAM subsets.⁴¹

As mentioned above, TAMs support cancer cell invasion and migration, which can be achieved by regulating genes related to metastasis. Hence, many TAM identification markers can be used as metastatic markers in various tumors models and experimental systems.⁴⁷ For example, invasion and migration of cancer cells can be studied by assessing the levels of CD11b⁺/Gr1^mid/lowTAMs in MC38 and LLC tumor models in mouse in vitro,⁴⁸ F4/80⁺ and/or CD11b⁺Gr1-F4/80⁺ TAMs in MMTV-PyMT in mouse in vivo,^49,50 CD68⁺ CD163⁺ in THP-1 and in human colorectal cancer samples.⁵¹ Moreover, CSF-1R⁺TAMs in hepatocellular carcinoma were related to increased intrahepatic metastasis, tumor recurrence, and reduced patient survival.⁵²

Previous studies have reported that TAMs express Programmed cell death protein 1 (PD-1)⁵³ and Programmed cell death ligand 1 (PD-L1)^54,55 within the TME. PD-1 is an immune checkpoint receptor upregulated in activated T cells for the induction of immune tolerance. Tumor cells frequently overexpress PD-L1, thus facilitating immune escape.⁵⁶ Mouse and human TAMs expressing PD-1 (herein, TAM PD-1) are negatively correlated with phagocytic potency against tumor cells, and the blockade of PD-1/PD-L1 receptors in vivo increases phagocytosis, reduces tumor growth, and prolongs survival of cancer mouse models in a Mφ-dependent manner.⁵³ Several recent studies have suggested that one of the immunosuppressive functions of TAM is the regulation of PD-L1, which is expressed on TAM in many cancer types, and stimulation of the apoptosis of T cells via interaction with PD-1 on CD8+T cells.^54,57

Therefore, we aimed to investigate the application of MAFB as a suitable biomarker for the TAM PD-1⁺ and/or TAM PD-L1⁺, for use as a target for cancer.

Immunostaining of human cancer tissues

We analyzed cancer tissues of human patients (n = 144) obtained from the University of Tsukuba Hospital. Frozen human lung, liver, colon, and pancreatic tumor tissues were sectioned (5 μm), paraffin-embedded, and visualized as previously described¹ using 1:100 anti-CD68 (clone SQab1742; Arigo Biolaboratories, Hsinchu, Taiwan, ROC), 1:100 anti-CD204 (SRA-E5; Trans Genic, Tokyo, Japan), 1:50 anti-MAFB (clone OTI2A6; Lifespans Biosciences, Seattle, WA, USA), 1:50 anti-PD-1 (clone NAT105; Abcam, Cambridge, UK), and 1:50 anti PD-L1 (clone SP142; clone 28-8; Abcam, Cambridge, UK) antibodies. Positively stained areas were quantified using BZ-X800 analyzer (Keyence, Itasca, Illinois, U.S.A.). All samples were obtained with informed consent and in accordance with the ethics committee of the University of Tsukuba Hospital (approval number H30-66).

Animals

Mice with C57BL/6J background raised in our center (Lab Animal Resource Center, The University of Tsukuba, Japan) were used. To generate MafB conditional knockout (cKO) mice, the Mafb gene flanked by a loxP constituent were used.⁵⁸ Subsequently, these mice were crossed with Lysm-Cre knock-in mice to obtain (Mafb^flox/flox::Lysm-Cre) and the control WT (wild type) and (Mafb^flox/flox) animals. Mafb GFP knock-in mice (Mafb heterozygous (Mafb^gfp/+) were created by inserting green fluorescent protein (GFP) gene at the Mafb locus as previously described.³ Genotypes were verified by analyzing the genomic DNA extracted from mouse tail tips. The primer sequences used were as previously reported.¹²

Mice were maintained under specific pathogen-free conditions at the Laboratory Animal Resource Center, University of Tsukuba, Japan. All experiments were performed in compliance with Japanese institutional laws and guidelines and were approved by the University of Tsukuba Animal Ethics Committee (approval number 19-141).

Cell culture

LLC cells were cultured in a 58 cm² cell culture dish (Greiner Bio-One) containing the nutrient medium, Dulbecco’s modified Eagle’s medium (DMEM, Sigma) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Gibco) and incubated in a humidified incubator (37 °C and 5% CO2).

Fluorescence-Activated Cell Sorting (FACS) analysis

LLC tumors were injected into WT, Mafb^gfp/+, and Mafb^flox/flox::Lysm-Cre mice by the subcutaneous injection (s/c) of LLC cells (approximately 2 × 10⁵ cells) in the right flank. After 21 days, the mice were dissected, and LLC tumors were harvested, weighed, placed in PBS, and sectioned into small pieces using sharp blades. Next, the tumor sections were incubated at 37 °C for 30 min in Roswell Park Memorial Institute medium (RPMI medium, Sigma) containing 2% collagenase A (Nitta Gelatin Inc.) and 1% DNase I (Roche). After gentle trituration, cells were separated and filtered through a 100 μm cell strainer (BD Bioscience). These cells were immunostained using antibodies against F4/80 (AbD Serotec), CD204 (AbD Serotec), PD-1 (Biolegend), and PD-L1 (Biolegend) and stained with 4ʹ,6-diamidino-2-phenylindole (DAPI) (Dojindo). All cells were analyzed using the BD LSR (BD Bioscience).

Statistical analysis

Data are expressed as the mean  ± S.E.M and analyzed using Welch’s t-test. The Kaplan–Meier method was used to estimate overall survival time; the difference in survival was compared by log-rank test, the two paired groups by Wilcoxon test, and the different survival distributions by Tarone-Ware test. In survival analysis, we adjusted the p-value by Dunn-Sidak correction. Based on patient data that we received from the hospital of the University of Tsukuba, TNM staging system was used to classify the severity and the extent of spread of cancer of all human cancer patients who participated in this study. Differences were considered statistically significant at p  < 0.05.

MAFB was expressed in various human cancers

To confirm that MAFB is indeed expressed in different human cancer types, we conducted an immunohistochemical analysis of 130 lung adenocarcinoma, 6 colon cancer, 6 liver cancer, and 2 pancreatic cancer samples using human anti-MAFB antibodies which confirmed that MAFB was expressed in all cases (Fig. 1).

MAFB level was significantly increased in lung adenocarcinoma showing metastasis potential

By performing immunohistochemical analysis of 2 groups of human lung adenocarcinoma samples, group one showed no tendency for metastasis (Metastasis -) (n = 60) (Fig. 2A) and group 2 showed a tendency for metastasis (Metastasis +) (n = 60) (Fig. 2B). By the morphological quantification relative to the total tissue area, we confirmed that MAFB expression was significantly increased in lung adenocarcinoma showing metastatic potential (Supplementary Fig. 3 and Fig. 2C).

Moreover, we confirmed that the increased MAFB expression in group 2 compared to group one was related to poor survival rate (Fig. 2D). These findings indicate that increased MAFB expression in human lung adenocarcinoma was related to increased tendency for metastasis and thus, is a prognostic indictor for increased cancer risk and poor survival rate.

MAFB expression was significantly increased in severe colon cancers simultaneously with that of CD204 and CD68

We compared the expression of MAFB and other common malignancy markers for TAMs. Hence, we performed immunohistochemical analysis on 2 groups of human colon cancer samples, with group one representing the mild cases (n = 3) and group 2 representing the severe cases (n = 3), using human anti-MAFB, human anti-CD204, and human anti-CD68 antibodies. We found that MAFB was expressed at nearly the same expression sites of CD204 and CD68 markers in both the mild cases (Fig. 3A) and severe cases (Fig. 3B). Furthermore, MAFB was specifically expressed in the nuclei of Mφs, while CD204 and CD68 were expressed on the outer cell surfaces of many cells within the TME in the mild cases (Fig. 3C, E) and severe cases (Fig. 3D, F), respectively. However, by using CD204 and CD68 only, it was difficult to distinguish the Mφs from other cells.

Then, we performed morphological quantification of the MAFB, CD204, and CD68 expression relative to the total tissue area, and we confirmed that MAFB expression significantly increased simultaneously with the increase in expression of CD204 and CD68 in group 2 representing the severe colon cancer cases (Supplementary Fig. 1).

MAFB expression was significantly increased in severe liver cancers simultaneously with that of CD204 and CD68

We performed immunohistochemical analysis using samples of 2 groups of human liver cancers; group one represented the mild cases (n = 3) and group 2 represented the severe cases (n = 3). We used human anti-MAFB, human anti-CD204, and human anti-CD68 antibodies. We confirmed that MAFB was expressed at nearly the same expression sites of CD204 and CD68 markers in both mild cases (Fig. 4A) and severe cases (Fig. 4B). Similar to the result in colon cancer samples, MAFB was specifically expressed in the nuclei of Mφs, while CD204 and CD68 were expressed on the outer cell surfaces of many cells within the tumor tissues in the mild cases (Fig. 4C, E) and severe cases (Fig. 4D, F), respectively.

We also performed morphological quantification of MAFB, CD204, and CD68 expression relative to the total tissue area, and similar to the previous result, MAFB expression was significantly increased simultaneously with the increase in the expression of CD204 and CD68 in group 2 representing the severe liver cancer cases (Supplementary Fig. 2).

MAFB was expressed in severe pancreatic cancers

Finally, we performed immunohistochemical analysis using samples of a group of patients with severe pancreatic cancer (n = 2) using human anti-MAFB (Fig. 5A), human Anti-CD204 (Fig. 5B), and human anti-CD68 (Fig. 5C) antibodies. As with the previous results for colon and liver cancers, we found that MAFB was specifically expressed in the nuclei of Mφs, while CD204 and CD68 were expressed on the outer cell surfaces of many other cells within the TME.

GFP-MAFB signals were expressed within the PD-1 or/and PDL-1 positive population in Lewis lung carcinoma (LLC) mouse model

We hypothesized that MAFB could be a suitable target for drug development targeting TAM PD-1⁺ and/or TAM PDL-1⁺ cells. Hence, we performed FACS analysis of 3-weeks-old LLC tumors collected from wild type mice (n = 5), and Mafb^gfp/+ mice (n = 5). Then we checked the expression of GFP, F4/80, PD-1, and PD-L1 after gating the viable cells. Our results showed that GFP signals, indicating MAFB expression, were observed within the PD-1⁺ or PDL-1⁺ populations separately (Fig. 6A) and also in about 35.3% of the double-positive TAM PD-1⁺/PD-L1⁺ population (Fig. 6B).

MAFB was expressed in TAM PD-1⁺ or/and TAM PDL-1⁺ cells but did not regulate their expression in LLC mouse model

To confirm whether MAFB expressed in TAM can regulate the PD-1 or PD-L1 expression, we injected LLC cells into a group of control mice (Mafb^flox/flox) (n = 5) and a group of Mafb^flox/flox::Lysm-Cre mice (n = 5). After 3 weeks, LLC tumors were collected from the 2 groups, and the expression of F4/80, CD204, PD-1, and PD-L1 were assessed after gating the viable cells. We found that there was no significant difference in the expression of PD-1 and PD-L1 between the WT group (Fig. 7A) and MAFB-conditional knockout group (Fig. 7B). These findings suggest that MAFB is expressed in TAM PD-1⁺ or/and TAM PD-L1⁺ cells but does not regulate their expression.

To confirm MAFB expression within PD-1 and PD-L1 populations in human cancers, we performed immunohistochemical analysis on cancer tissues collected from a group of patients with lung adenocarcinoma (n = 5) using human anti-MAFB, human anti-PD-1, and human anti-PD-L1 antibodies. We confirmed that MAFB (Fig. 8B) was nearly co-expressed in the same tissue area with PD-1⁺ (Fig. 8C) and/or PD-L1⁺ (Fig. 8D). These results indicate that MAFB can be suitable marker for TAM PD-1⁺ or/and PD-L1⁺ cells in lung adenocarcinoma and consequently, it could be a suitable target for cancer immunotherapy.

Previously, we have shown that MAFB could be a suitable marker for TAMs in human lung adenocarcinoma and in LLC mouse models.¹ In this study, we proved that the transcription factor MAFB could be a biomarker for various human cancers including hepatic, colon, and pancreatic cancers in addition to our previous findings. Moreover, in this study, we first showed that high expression of MAFB was correlated to increased metastatic potential of cancer cells in patients with lung adenocarcinoma. In addition, high MAFB expression was associated with increased risk and poor survival rate among these patients.

Additionally, our results harmonize with the previous studies that stated that MAFB could have regulatory roles in hepatocellular carcinoma,⁵⁹ colorectal cancers,⁶⁰ and pancreatic ductal adenocarcinoma,⁶¹ but we first showed that high MAFB expression in these cancers was closely related with increased severity and poor prognosis.

Several markers can be used to mark M2 type TAMs within the TME in both cancer samples or any other experimental systems; these markers include CD68, CD204, CD136, CD11b, F4/80, and CSF-1R. ^42,45–52 However, in clinical practice, these markers have diverse expression levels that indicate distinct TAM phenotypes and are correlated with different prognostic values. Some cells within TME or M1 TAM can express some of these markers as well.^{36–38,41,43,44} We showed that transcriptional factor MAFB was expressed specifically in M2 TAMs in various human cancers and murine tumor models and was highly expressed within the TAM nuclei making their morphological quantification easier. Furthermore, high MAFB expression within the studied cancers correlated with overall poor prognostic values.

Lastly, we showed that MAFB was expressed in TAM PD⁺ and/or TAM PDL-1⁺ cell populations in both human lung adenocarcinoma and LLC mouse models which agrees with the previous studies.^53–55 This finding indicates that MAFB can be suitable marker for TAM PD-1+ or/and PD-L1+ population within lung adenocarcinoma TME and accordingly it could be a suitable target for cancer immunotherapies targeting these cell populations.

In conclusion, we suggest the potential of transcription factor MAFB as a biomarker for many human cancers and murine tumor models. In our more detailed studies on human lung adenocarcinoma, MAFB can be a marker for many prognostic values including tumor severity, cancer cell migration, invasion, metastasis, and overall poor survival rates.

Finally, MAFB can a potential target for cancer immunotherapies. However, more studies should be conducted to determine the molecular roles of MAFB within TAMs and to detect the exact roles of MAFB in TAM PD⁺ and/or TAM PDL-1⁺ cells in different human cancer types.

Acknowledgments

We want to thank our lab mates from Lab animal resource center at the University of Tsukuba, Japan, for providing insights and assisting in the research. We also wish to extend our appreciation to Tomoyo Takeuchi at Tsukuba Human Tissue Biobank Center, the University of Tsukuba, Japan, for obtaining the human tissue sections.

Author Contributions

O.S and M.H provided study concept and design; O.S, M.S, and M.H performed methodology and writing, review, and revision of the paper; O.S, M.S, M.K, N.K, and Y.I acquired human samples, analyzed and interpreted the data, and performed statistical analysis; S.T provided technical and material support. All authors read and approved the final paper.

Data Availability Statement

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Funding This work was funded by the Ministry of Education and Sports of Japan and supported by the Ministry of Education JSPS KAKENHI (grant numbers 26221004, 25860205, 23118504, 16K18398, 19K07499, and 19H00966), the Takamatsunomiya Cancer Foundation (grant number 15–24724; M. Hamada), the Culture, Sports, Science and Technology of Japan (MEXT), the Uehara Memorial Foundation, Takeda Science Foundation, and the World Premier International Research Center Initiative, MEXT, Japan. Corresponding authors: Michito Hamada, Ph.D., Department of Anatomy and Embryology, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8575, Japan; Phone: +81-298-53-7516, Fax: +81-298-53-6965, E-mail: [email protected]

Satoru Takahashi, Ph.D., Department of Anatomy and Embryology, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8575, Japan; Phone: +81-298-53-7516, Fax: +81-298-53-6965, E-mail: [email protected] Competing Interests: The authors declare that there is no conflict of interest. Word count of the manuscript: 3712 excluding references and figure legends Number of figures: 8 figures and 3 supplementary figures

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There is NO conflict of interest to disclose.

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MAFB is a biomarker for cancer severity and prognosis, and is a potential cancer immunotherapy target

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